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. 2012 Nov 30;287(49):41374-85.
doi: 10.1074/jbc.M112.400044. Epub 2012 Oct 5.

Absence of glial α-dystrobrevin causes abnormalities of the blood-brain barrier and progressive brain edema

Chun Fu Lien  1 , Sarajo Kumar Mohanta, Malgorzata Frontczak-Baniewicz, Jerome D Swinny, Barbara Zablocka, Dariusz C Górecki
Affiliations

Absence of glial α-dystrobrevin causes abnormalities of the blood-brain barrier and progressive brain edema

Chun Fu Lien et al. J Biol Chem. .

Abstract

The blood-brain barrier (BBB) plays a key role in maintaining brain functionality. Although mammalian BBB is formed by endothelial cells, its function requires interactions between endotheliocytes and glia. To understand the molecular mechanisms involved in these interactions is currently a major challenge. We show here that α-dystrobrevin (α-DB), a protein contributing to dystrophin-associated protein scaffolds in astrocytic endfeet, is essential for the formation and functioning of BBB. The absence of α-DB in null brains resulted in abnormal brain capillary permeability, progressively escalating brain edema, and damage of the neurovascular unit. Analyses in situ and in two-dimensional and three-dimensional in vitro models of BBB containing α-DB-null astrocytes demonstrated these abnormalities to be associated with loss of aquaporin-4 water and Kir4.1 potassium channels from glial endfeet, formation of intracellular vacuoles in α-DB-null astrocytes, and defects of the astrocyte-endothelial interactions. These caused deregulation of tight junction proteins in the endothelia. Importantly, α-DB but not dystrophins showed continuous expression throughout development in BBB models. Thus, α-DB emerges as a central organizer of dystrophin-associated protein in glial endfeet and a rare example of a glial protein with a role in maintaining BBB function. Its abnormalities might therefore lead to BBB dysfunction.

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Figures

FIGURE 1.
FIGURE 1.
Leaky BBB in α-dystrobrevin knock-out (ADB) brains. A, macroscopic views of wild-type, ADB, and mdxβgeo (positive control) brains showing Evans blue extravasation in specific brain areas. B, confocal micrographs showing Evans blue extravasations (yellow fluorescence) into the parenchyma of ADB and mdxβgeo brains. C, confocal micrographs of blood vessels in the wild-type and ADB brain cortices stained for fibrinogen (top), IgG (middle), and albumin (bottom). Immunoreactivity for these blood components (red) is present outside the GS-IB4-labeled (green) blood vessels. D, quantitative evaluation of fibrinogen and IgG immunoreactivities expressed as number of positive blood vessels (BVs) per area (mm2) of cortex showed highly statistically significant differences (*, p < 0.001) between wild-type and ADB brains. Error bars represent ± S.E.
FIGURE 2.
FIGURE 2.
Structural and functional abnormalities in endothelia-ADB astrocyte co-cultures. A, phase-contrast images of morphological developments in BEC-astrocyte co-cultures. Panel 1, confluent monolayer of wild-type primary astrocytes (caret) is penetrated by BEC added into the culture (panel 2). The presence of BEC (asterisk) triggers rearrangement of glia into islands (panel 2, the arrows denote astrocytic island margins) interconnected by thick multicellular glial columns. The size of the islands decreases with time in culture (panel 3), whereas the columns elongate and the complexity of their network increases. B, comparison of phase-contrast images of BEC co-cultured with wild-type or ADB astrocytes. Control astrocytes (panels a and b) formed typical arrangements, which were highly reproducible and stable. The ADB glia lattice framework was significantly less complex and fragile (panels c and d) as shown by statistically significant differences (p < 0.01) in morphometric analyses (C) where ADB columns were longer and the surface areas in between lattices larger. D, normalized ECIS plot (percentage of relative resistance versus time) showing endothelial resistance development in BEC cultured on wild-type or ADB astrocyte-derived ECMs (i.e. no live cell contact). E, relative resistance in BEC co-cultured with either wild-type or ADB astrocytes (live cell-cell contact). Note the significant divergence in ECIS values between cultures containing the two astrocyte types but no difference when glia-derived ECM were used.
FIGURE 3.
FIGURE 3.
Alterations at glial endfeet in the absence of α-dystrobrevin. Representative confocal immunofluorescence images were taken under identical settings through glia in BEC/astrocyte co-cultures. A and B, in ADB co-cultures, analyses revealed vacuolated spaces (arrowheads) near the cell attachment points on the basal surface. Staining for Kir4.1 (A, green) and AQP4 (B, green) revealed significantly decreased expression levels and disruption of normal co-localization of these proteins with β-dystroglycan (β-DG, red) of DAP complexes. Note the diffuse distribution and loss of co-localization (yellow signal, arrows) in ADB samples (A and B). This coincided with disruption of the DAP assembly itself in the absence of α-DB (C) as shown here by the lack of signal co-localization for β-dystroglycan and its extracellular ligand, laminin (D).
FIGURE 4.
FIGURE 4.
Malformation of three-dimensional assemblies of BEC co-cultured with ADB glia. Wild-type astrocytes induced the formation of well delineated tubular endothelial structures clearly evident at day 4. Confocal microscopy analyses at day 6 confirmed that GFAP-labeled wild-type astrocytes were in contact with the GS-IB4-labeled endothelial tubes, whereas ADB astrocytes did not support the endothelial tubulogenesis. Here, remnants of cyst-like structures that initiated the early branching of endothelial tubes were still fairly intact at days 4 and 6. Although contacts between ADB astrocytes and endothelial tube-like structures were made, the latter appeared unusually thick. Left-side panels, phase-contrast images of three-dimensional cultures of glia with BEC. In the WT panel, note the fine, dense connections with intricate vessel-like structures at day 4 in vitro (DIV 4), becoming more pronounced and networked by day 6. In the ADB panel, note fewer, broader, and discontinuous structures and cysts (arrows), a sign that early vessel development is evident. By day 6, some cysts remained, and vessel assemblies broadened. Right-hand side panels, representative confocal images in three-dimensional co-cultures. BEC and wild-type astrocytes formed well defined tubular structures, with astrocytes (positive for GFAP, green) arranged in a continuous sheath along and around the endothelial vessel-like assemblies (stained with GS-IB4, red). In the presence of ADB glia, these orderly arrangements were lost, and cells have grown in large, dense clusters (arrows).
FIGURE 5.
FIGURE 5.
Abnormalities in expression and assembly patterns of glial and endothelial proteins in ADB co-cultures. A, representative immunoblots of protein extracts are shown. In solo cultures, BEC cultured alone expressed ZO-1, occludin (OCLN), low levels of β-DB, dystroglycans (DG), syntrophins (SYN), and laminin (LAM). In astrocytes alone, dystrobrevins, dystroglycans, syntrophins, dystrophin Dp71, and ZO-1 were found. Molecular mass (kDa) of each protein is indicated. Actin was used as a control for equal protein loading. Immunoblots of protein extracts from co-cultures of BEC with WT or ADB astrocytes over 8 days in culture show dystrophin and DAPs, DAP-interacting proteins, and BBB markers. There were significant delays in temporal expression patterns and in levels of expression of AQP4, laminin, and Kir4.1 in ADB co-cultures. Expression of dystrophin Dp71 was lower in ADB, and the time course analysis showed gradual decrease of Dp71 levels from day 4 onwards in both control and ADB samples. Expression of BBB marker occludin was detectable from day 2 in wild-type cultures, but in ADB samples, it was noticeable from day 6 only. B and C, blue native-PAGE analysis of OAP formation. AQP4 immunoblots following blue native-PAGE of cell lysates of wild-type and ADB astrocytes either pretreated with increasing concentrations of laminin-1 (20 and 40 nm) (B) or co-cultured with BEC (C). Under both conditions, ADB astrocytes failed to form OAP, in clear contrast to the wild-type cells.
FIGURE 6.
FIGURE 6.
Abnormal localization of Kir4.1 and AQP4 in ADB brains. A and B, representative micrographs of immunolocalizations from AQP4 (A) and Kir4.1 (B) in mouse brains. A1, in the small magnification view of hippocampus, AQP4 staining (green) delineates capillaries in the wild-type brains but not in ADB brains. Confocal co-localizations of AQP4 (A2, green) and Kir4.1 (B, green) with GS-IB4-labeled endothelia (A2 and B, red) show AQP4 and Kir4.1 signals located in close proximity to endothelia (yellow, arrows) in WT but not ADB brains. Inset, Larger magnification image confirms that this corresponds with the loss of signals from ADB astrocytic endfeet (arrows). Hoechst (blue/white) was used as a nuclear counterstain, where indicated. C, representative immunoblots of brain protein extracts demonstrate the relative levels of AQP4, Kir4.1, syntrophin (SYN), and β-DB in the absence of α-DB. Actin was used as a control for equal protein loading. D, total brain mass and base-line water content analyses showed significant differences (p < 0.005 and p < 0.0005) between age-matched 3-month-old WT and ADB brains.
FIGURE 7.
FIGURE 7.
Absence of α-DB causes spongiform neurodegeneration and progressive brain edema. A, H&E staining of hippocampal areas of 18-month-old WT and ADB mice showing massive spongiosis evident in the parenchyma of the pyramidal cell layer, particularly regions CA2 and CA3, and in the granule cell layer of the dentate gyrus (DG) in ADB brains. B, electron microscopic images of brain cortex of WT (panel a) and ADB (panel b) mice. Panel a, regular capillary vessel (V) with pericytes (P) and endothelium (E) characterized by necrotic features in some cells (en), typically associated with aging. Panel b, capillary vessel in ADB cortex surrounded by vast astrocytic edema (asterisk). Endothelial cell cytoplasm is rich in pinocytotic vesicles. The arrow points to an improper tight junction between endothelial cells.
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