Hartwig Wolburg

Tight junctions of the blood–brain barrier: development, composition and regulation

1. The blood-brain barrier is essential for the maintenance and regulation of the neural microenvironment. The main characteristic features of blood-brain barrier endothelial cells are an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. 2. Endothelial blood-brain barrier tight junctions differ from epithelial tight junctions, not only by distinct morphological and molecular properties, but also by the fact that endothelial tight junctions are more sensitive to microenvironmental than epithelial factors. 3. Many ubiquitous molecular tight junction components have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin and 7H6. Signaling pathways involved in tight junction regulation include G-proteins, serine-, threonine- and tyrosine-kinases, extra and intracellular calcium levels, cAMP levels, proteases and cytokines. Common to most of these pathways is the modulation of cytoskeletal elements and the connection of tight junction transmembrane molecules to the cytoskeleton. Additionally, crosstalk between components of the tight junction- and the cadherin-catenin system of the adherens junction suggests a close functional interdependence of the two cell-cell contact systems. 4. Important new molecular aspects of tight junction regulation were recently elucidated. This review provides an integration of these new results.

Development of the blood-brain barrier

The microenvironment of the CNS is important for neuronal function, and the blood-brain barrier is involved in its maintenance. The barrier is present in a complex cellular system at the level of the tight junctions between endothelial cells. The unique properties of the endothelial cells in the CNS compared with those present in other organs are not predetermined by brain-specific endothelial precursors but are induced by the neural environment during the development of the vascular system. Astrocytes that tightly appose endfeet onto the abluminal side of brain capillaries seem to be important for the induction and maintenance of the endothelial barrier.

Aβ42‐driven cerebral amyloidosis in transgenic mice reveals early and robust pathology

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron‐specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6–8 weeks and the ratio of human amyloid (A)β42 to Aβ40 is 1.5 and 5 in pre‐depositing and amyloid‐depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid‐associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau‐positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross‐breeding to other genetically engineered mouse models.

Tight junctions of the blood-brain barrier.

Abstract1. The blood–brain barrier is essential for the maintainance and regulation of the neural microenvironment. The blood–brain barrier endothelial cells comprise an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. The latter is realized by the tight junctions between the endothelial cells of the brain microvasculature, which are subject of this review. Morphologically, blood–brain barrier-tight junctions are more similar to epithelial tight junctions than to endothelial tight junctions in peripheral blood vessels.2. Although blood–brain barrier-tight junctions share many characteristics with epithelial tight junctions, there are also essential differences. However, in contrast to tight junctions in epithelial systems, structural and functional characteristics of tight junctions in endothelial cells are highly sensitive to ambient factors.3. Many ubiquitous molecular constituents of tight junctions have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin, and 7H6. Signaling pathways involved in tight junction regulation comprise, among others, G-proteins, serine, threonine, and tyrosine kinases, extra- and intracellular calcium levels, cAMP levels, proteases, and TNFα. Common to most of these pathways is the modulation of cytoskeletal elements which may define blood–brain barrier characteristics. Additionally, cross-talk between components of the tight junction– and the cadherin–catenin system suggests a close functional interdependence of the two cell–cell contact systems.4. Recent studies were able to elucidate crucial aspects of the molecular basis of tight junction regulation. An integration of new results into previous morphological work is the central intention of this review.

Wnt/β-catenin signaling controls development of the blood–brain barrier

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.

Viscoelastic properties of individual glial cells and neurons in the CNS

One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word “γλια” means “glue”) or provide a robust scaffold for them (“support cells”). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft (“rubber elastic”), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.

Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme

Abstract The aim of the study was to characterize the interendothelial junctions in tumor microvessels of five cases of human glioblastoma multiforme. In addition to morphological analysis, tumors were screened for the expression of junctional proteins, such as occludin, claudin-1, ZO-1 and catenins. The expression of the tight junction protein claudin-1 was lost in the majority of tumor microvessels, whereas claudin-5 and occludin were significantly down-regulated only in hyperplastic vessels. As shown by freeze-fracture analysis, under the conditions of tumor growth tight junction particles of endothelial cells were almost exclusively associated with the exocytoplasmic fracture face, providing evidence for a switch of the particles from the protoplasmic to the external leaflet of the endothelial membrane. These results suggest a relationship between claudin-1 suppression and the alteration of tight junction morphology, which is likely to correlate with the increase of endothelial permeability. Underlining the undifferentiated state of tumor microvessels, plakoglobin, a crucial protein for mature endothelial junctions, was not detectable in most microvessels, whereas β-catenin was abundantly labeled. In this context, it is of particular interest that the majority of microvascular pericytes were negative for alpha-smooth muscle actin, which is a marker of differentiated pericytes, although pericytes were frequently found in electron micrographs. In conclusion, the data suggest that the increase in microvascular permeability in human gliomas, contributing to the clinically severe symptoms of brain edema, is a result of a dysregulation of junctional proteins.

Localization of claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme.

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). During inflammation, BBB properties are frequently lost, resulting in brain edema. To investigate whether BBB leakiness correlates with molecular changes at BBB TJs, we performed immunofluorescence stainings for TJ molecules in a mouse model of experimental autoimmune encephalomyelitis (EAE) and in human tissue with glioblastoma multiforme (GBM). In TJs of healthy CNS vessels in both mouse and man we detected occludin, ZO-1, claudin-5 and claudin-3. In EAE brain and spinal cord sections we observed the selective loss of claudin-3 immunostaining from TJs of venules surrounded by inflammatory cuffs, whereas the localization of the other TJ proteins remained unchanged. In addition, selective loss of claudin-3 immunostaining was also observed in altered cerebral microvessels of human GBM. Our data demonstrate the selective loss of claudin-3 from BBB TJs under pathological conditions such as EAE or GBM when the integrity of the BBB is compromised, and therefore suggest that claudin-3 is a central component determining the integrity of BBB TJs in vivo.

Formation and maintenance of Alzheimer's disease beta-amyloid plaques in the absence of microglia

In Alzheimers disease, microglia cluster around β-amyloid deposits, suggesting that these cells are important for amyloid plaque formation, maintenance and/or clearance. We crossed two distinct APP transgenic mouse strains with CD11b-HSVTK mice, in which nearly complete ablation of microglia was achieved for up to 4 weeks after ganciclovir application. Neither amyloid plaque formation and maintenance nor amyloid-associated neuritic dystrophy depended on the presence of microglia.

Mini-review: Transendothelial migration of leukocytes: through the front door or around the side of the house?

Leukocyte adhesion to the endothelial cells lining the vessel wall and the subsequent migration of the leukocytes into the underlying tissue are key elements of both innate and adaptive immunity. Leukocyte extravasation is generally believed to take place through small gaps at intercellular endothelial cell junctions – the paracellular route. This view has, however, been repeatedly challenged by morphological studies demonstrating leukocyte migration through the endothelial cells themselves – the transcellular pathway. On the basis of the current experimental evidence, we propose consideration that both pathways are equally possible for a leukocytes journey from the apical surface of the endothelium to its basal side.