Astrocyte mediated modulation of blood-brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins β-catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ protein

An Allograft Glioma Model Reveals the Dependence of Aquaporin-4 Expression on the Brain Microenvironment

Aquaporin-4 (AQP4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. In contrast, most cultivated glioma cell lines don’t express AQP4, and primary cell cultures of human glioblastoma lose it during the first passages. Accordingly, in C6 cells and RG2 cells, two glioma cell lines of the rat, and in SMA mouse glioma cell lines, we found no AQP4 expression. We confirmed an AQP4 loss in primary human glioblastoma cell cultures after a few passages. RG-2 glioma cells if grafted into the brain developed AQP4 expression. This led us consider the possibility of AQP4 expression depends on brain microenvironment. In previous studies, we observed that the typical morphological conformation of AQP4 as orthogonal arrays of particles (OAP) depended on the extracellular matrix component agrin. In this study, we showed for the first time implanted AQP4 negative glioma cells in animal brain or flank to express AQP4 specifically in the intracerebral gliomas but neither in the extracranial nor in the flank gliomas. AQP4 expression in intracerebral gliomas went along with an OAP loss, compared to normal brain tissue. AQP4 staining in vivo normally is polarized in the astrocytic endfoot membranes at the glia limitans superficialis and perivascularis, but in C6 and RG2 tumors the AQP4 staining is redistributed over the whole glioma cell as in human glioblastoma. In contrast, primary rat or mouse astrocytes in culture did not lose their ability to express AQP4, and they were able to form few OAPs

Loss of astrocyte polarity marks blood-brain barrier impairment during experimental autoimmune encephalomyelitis

In multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE), dysfunction of the blood-brain barrier (BBB) leads to edema formation within the central nervous system. The molecular mechanisms of edema formation in EAE/MS are poorly understood. We hypothesized that edema formation is due to imbalanced water transport across the BBB caused by a disturbed crosstalk between BBB endothelium and astrocytes. Here, we demonstrate at the light microscopic and ultrastructural level, the loss of polarized localization of the water channel protein aquaporin-4 (AQP4) in astrocytic endfeet surrounding microvessels during EAE. AQP4 was found to be redistributed over the entire astrocytic cell surface and lost its arrangement in orthogonal arrays of intramembranous particles as seen in the freeze-fracture replica. In addition, immunostaining for the astrocytic extracellular matrix receptor beta-dystroglycan disappeared from astroglial membranes in the vicinity of inflammatory cuffs, whereas immunostaining for the dystroglycan ligands agrin and laminin in the perivascular basement membrane remained unchanged. Our data suggest that during EAE, loss of beta-dystroglycan-mediated astrocyte foot process anchoring to the basement membrane leads to loss of polarized AQP4 localization in astrocytic endfeet, and thus to edema formation in EAE

Freeze fracture analysis of the C6 system.

<p>(A) C6 implanted intracerebral tumor, (B), C6 cell culture. C6 cells implanted extracranial (C) and flank tumor (D). There are no OAPs. In contrast (BB) shows a freeze fracture replica of primary astrocytes in culture revealing a few OAPs (encircled). Scale bars 100 nm.</p

List of primer sequences used in this study.

<p>List of primer sequences used in this study.</p

Immunoreactivity against AQP4.

<p>(A) Rat control brain, AQP4 (red) is restricted to astrocytic endfeet contacting the blood vessel (green ZO-1). (B) Extracranial implanted C6-tumor, no AQP4 could be detected, green ZO-1. (C) C6-tumor implanted into the flank, no AQP4 could be detected, green ZO-1. (D) Intracerebral implanted C6-tumor, immunofluorescence (red) shows an intensive staining for AQP4 (L: blood vessel lumen). (E) Right side: Intracerebral implanted RG-2-tumor (T); AQP4 (red) is present in astrocytes all over the tumor tissue and stronger fluorescence in the reactive astrocytes, compared to the healthy part of the brain (N), GFAP (green). Left side: healthy part of the brain (N), AQP4 (red) is restricted to astrocytic membranes contacting the blood vessels. (F) No AQP4 could be detected in C6 cell cultures. Scale bars each 20 µm.</p

Immunoreactivity against AQP4 (red) and GFAP (green).

<p>(A) In human glioblastoma tissue AQP4 (red) shows an intensive staining whereas in primary cell culture of this glioblastoma AQP4 could not be detected (B). (C) Primary cell culture of astrocytes stained for AQP4. (D) Immunoblot against AQP4; the lower band (32 kDA) represents the AQP4 isoform M23 and the upper band (34 kDA) the isoform M1. Tubulin was used as loading control for these samples (40 kDA). The western blot is positive for AQP4 in the glioblastoma tissue (left lane), whereas the primary glioma cell cultures were negative (middle). Primary mouse astrocytes are positive for AQP4 (right lane). The M23-AQP4 isoform always shows a stronger band than the M1 isoform.</p

Freeze fracture analysis.

<p>(A) Normal human brain tissue and rat brain tissue (D) showing astrocytic endfoot membranes studded with OAPs containing AQP4. (B) Altered morphology of clustered OAPs in human glioblastoma tissue. (C) Primary cell culture membranes of human glioblastoma are devoid of OAPs as well as intracerebral RG-2 tumor (E), and the RG-2 cell line (F). Scale bars 100 nm.</p