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. 2008 Apr;28(4):812-23.
doi: 10.1038/sj.jcbfm.9600585. Epub 2007 Nov 21.

The rapid decrease in astrocyte-associated dystroglycan expression by focal cerebral ischemia is protease-dependent

Richard Milner  1 , Stephanie Hung, Xiaoyun Wang, Maria Spatz, Gregory J del Zoppo
Affiliations

The rapid decrease in astrocyte-associated dystroglycan expression by focal cerebral ischemia is protease-dependent

Richard Milner et al. J Cereb Blood Flow Metab. 2008 Apr.

Abstract

During focal cerebral ischemia, the detachment of astrocytes from the microvascular basal lamina is not completely explained by known integrin receptor expression changes. Here, the impact of experimental ischemia (oxygen-glucose deprivation (OGD)) on dystroglycan expression by murine endothelial cells and astrocytes grown on vascular matrix laminin, perlecan, or collagen and the impact of middle cerebral artery occlusion on alphabeta-dystroglycan within cerebral microvessels of the nonhuman primate were examined. Dystroglycan was expressed on all cerebral microvessels in cortical gray and white matter, and the striatum. Astrocyte adhesion to basal lamina proteins was managed in part by alpha-dystroglycan, while ischemia significantly reduced expression of dystroglycan both in vivo and in vitro. Furthermore, dystroglycan and integrin alpha6beta4 expressions on astrocyte end-feet decreased in parallel both in vivo and in vitro. The rapid loss of astrocyte dystroglycan during OGD appears protease-dependent, involving an matrix metalloproteinase-like activity. This may explain the rapid detachment of astrocytes from the microvascular basal lamina during ischemic injury, which could contribute to significant changes in microvascular integrity.

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Figures

Figure 1
Figure 1
Expression of α-dystroglycan (A and C) and β-dystroglycan (B and D) on normal microvessels within the striatum of the non-human primate (A and B) and C57Bl/6 mice (C and D). Scale bars = 100 μm (A and B) and 50 μm (C and D).
Figure 2
Figure 2
Expression of β-dystroglycan and the integrin β4 subunit relative to HSPG (perlecan) on select cerebral microvessels of the nonhuman primate in normoxic gray matter. Individual fluorescence channels of confocal microscopy are merged in (C), (F), (I), and (L). The expression of β-dystroglycan (B) appears outside HSPG (A), and subunit β4 (E) appears outside HSPG (D), in representative experiments. Both β-dystroglycan (H) and subunit β4 (G) are coexpressed by the same vascular structures. Generally, aquaporin-4 (J) and β-dystroglycan (K) appear to be colocalized to most microvessels in the gray matter. Scale bars = 50 μm.
Figure 3
Figure 3
Vascular coexpression of β-dystroglycan and the integrin β4 subunit in cortical gray matter (GM) and white matter (WM). Two representative fields (A to C and D to F) show the appearance of β-dystroglycan (C and F) in both GM and WM, but β4 (B and E) primarily in WM. Scale bar = 100 μm.
Figure 4
Figure 4
Microvessel distributions of β-dystroglycan and the integrin β4 subunit expression in the cortical gray matter (A) and white matter (B), and the striatum (C) of the non-human primate. β-Dystroglycan is found on all microvessels in the white and gray matter, whereas integrin β4 is on all microvessels in the white matter only (B). Solid line = β-dystroglycan, broken line = integrin subunit β4.
Figure 5
Figure 5
Expression of β-dystroglycan by cultured murine cerebral endothelial cells and astrocytes on collagen type IV (solid bar), HSPG/perlecan (hatched), and laminin (reverse hatched). For each matrix ligand, astrocytes expressed 3.3- to 4.1-fold more dystroglycan than endothelium (P < 0.0001 each). Inset: primary astrocytes contained both 51 and 31 kDa forms of β-dystroglycan, whereas endothelial cells expressed the 51 kDa form predominantly.
Figure 6
Figure 6
Inhibition of α-dystroglycan-mediated astrocyte–matrix adhesion. The anti-α-dystroglycan MoAb 11H6C4 significantly reduced astrocyte adhesion to laminin (P = 0.034; n = 3), but not to fibronectin.
Figure 7
Figure 7
A direct inverse correlation between the loss of the 51 kDa β-dystroglycan and the appearance of the 31 kDa form was observed in the striatum after 2 h MCAO and 7 days MCAO compared with nonischemic tissue (r = −0.7570, P = 0.009; n = 3 each). Inset: immunoblot of striatum at 0 h (lane 1), 2 h (lane 2), and 7 days (lane 3) after MCAO (see Materials and methods). Arrow = 31 kDa form.
Figure 8
Figure 8
β-Dystroglycan expression on astrocytes was significantly reduced by OGD when the cells were grown on collagen type IV (black), HSPG (fine hatched), or laminin (coarse hatched). A nonsignificant reduction in endothelial cell expression was seen (n = 3 each). Inset: no substantial evidence of cell demise (by propidium iodide) was observed between cells subject to OGD and normoxia.
Figure 9
Figure 9
Impact of OGD on astrocyte expression of β-dystroglycan protein and transcripts. Change in astrocyte β-dystroglycan content because of OGD when cells were grown on collagen IV (squares), HSPG (circles), or laminin (triangles), from immunoblots (n = 4 each) (A). OGD produced a significant increase in the appearance of the 31 kDa form of β-dystroglycan by astrocytes grown on collagen IV compared with normoxia (P = 0.046) (depicted are the ratios OGD/normoxia of each form; solid = 51 kDa form, hatched = 31 kDa form) (B). Increase in the relative transcription of β-dystroglycan mRNA induced in astrocytes grown on plastic and laminin, which were subject to OGD (2P = 0.007 and 0.006, respectively; n = 6 each) (ratio = OGD/normoxia) (C).
Figure 10
Figure 10
Preservation of β-dystroglycan expression by astrocytes exposed to various protease inhibitors (n = 3 each). Cells grown on collagen IV (A) or laminin (B) were maintained under normoxia (black bars) or exposed in parallel to OGD (hatched bars), in the absence (first data pair) or presence (subsequent pairs) of each protease inhibitor. OGD significantly decreased astrocyte β-dystroglycan expression relative to normoxia (collagen IV, P = 0.015; laminin, P < 0.01) for all inhibitors except GM6001 and 1,10-phenanthroline (P > 0.10 each).
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