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. 2012 Dec;60(12):2018-26.
doi: 10.1002/glia.22416. Epub 2012 Sep 17.

Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse

Rune Enger  1 , Georg Andreas Gundersen, Nadia Nabil Haj-Yasein, Martine Eilert-Olsen, Anna Elisabeth Thoren, Gry Fluge Vindedal, Pétur Henry Petersen, Øivind Skare, Maiken Nedergaard, Ole Petter Ottersen, Erlend A Nagelhus
Affiliations

Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse

Rune Enger et al. Glia. 2012 Dec.

Abstract

Key roles of macroglia are inextricably coupled to specialized membrane domains. The perivascular endfoot membrane has drawn particular attention, as this domain contains a unique complement of aquaporin-4 (AQP4) and other channel proteins that distinguishes it from perisynaptic membranes. Recent studies indicate that the polarization of macroglia is lost in a number of diseases, including temporal lobe epilepsy and Alzheimer's disease. A better understanding is required of the molecular underpinning of astroglial polarization, particularly when it comes to the significance of the dystrophin associated protein complex (DAPC). Here, we employ immunofluorescence and immunogold cytochemistry to analyze the molecular scaffolding in perivascular endfeet in macroglia of retina and three regions of brain (cortex, dentate gyrus, and cerebellum), using AQP4 as a marker. Compared with brain astrocytes, Müller cells (a class of retinal macroglia) exhibit lower densities of the scaffold proteins dystrophin and α-syntrophin (a DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or α-syntrophin--while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes--had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Müller cells. In addition, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of α-syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization.

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Figures

Figure 1
Figure 1
Immunofluorescence for dystrophin and α-syntrophin in cortex (A–D), dentate gyrus (EH), cerebellum (I–L) and retina (M–P). In cortex of wild-type mice strong immunofluorescence signals for dystrophin (A) and α-syntrophin (C) were seen along the pial surface (asterisks) and around cortical vessels (arrowheads). A similar pattern of immunolabeling was found in dentate gyrus (E,I) and cerebellum (G,K) for both antibodies. Insets: Co-immunostaining with the endothelial marker CD31 (blue) revealed that labeling for dystrophin and α-syntrophin was peripheral to the endothelium (arrows), corresponding to perivascular macroglial endfeet (arrowheads).The perivascular labeling for dystrophin (A,E,I,M) and α-syntrophin (C,G,K,O) was much weaker in retina than in the three regions of brain. Loss of immunolabeling in sections obtained from mdx3Cv (B,F,J,N) and α-syntrophin−/− mice (D,H,L,P) confirmed the selectivity of the anti-dystrophin and anti-α-syntrophin antibodies, respectively. Scale bars, 50 μm, 5 μm (insets).
Figure 2
Figure 2
Subcellular distribution of α-syntrophin in cortex and retina as revealed by immunogold cytochemistry. In wild-type mice gold particles signaling α-syntrophin decorate astrocytic endfoot membranes (arrowheads) peripheral to the endothelium (End) of cortical capillaries (A). The immunogold labeling of endfoot membranes (arrowheads) of retinal Müller cells abutting capillaries was very weak in both wild-type (C) and α-syntrophin−/− mice. B: Quantitative analysis of α-syntrophin labeling along perivascular macroglial endfoot membranes in cortex and retina. The ordinate shows number of gold particles per μm of membrane quantified. SEM, number of profiles (N), and p values for comparison are indicated. The α-syntrophin immunogold labeling of wild-type mice was significantly higher over perivascular endfoot membranes of cortical astrocytes than of retinal Müller cells. The latter immunosignal was, however, not significantly different from that of α-syntrophin−/− mice, indicating that it was indistinguishable from background labeling. Insets: Distribution of gold particles signaling α-syntrophin along an axis perpendicular to the perivascular endfoot membrane. The ordinate indicates number of gold particles per bin (bin width, 4 nm; cytoplasmic side negative). In cortex of wild-type mice a distinct peak is present corresponding to the plasma membrane and its cytoplasmic side, reflecting the localization of α-syntrophin. The gold particle density drops to background level ~30 nm from the midpoint of the membrane, corresponding to the size of the antibody bridge between the epitope and the corresponding gold particle. No distinct peak is seen over Müller cell endfoot membranes from wild-type and α-syntrophin−/− mice, indicating that the signal is unspecific labeling. Scale bar, 0.5 μm.
Figure 3
Figure 3
Distribution of AQP4 immunofluorescence in cortex (A–C), dentate gyrus (D–F), cerebellum (G–I) and retina (J–L) of wild-type (A,D,G,J), mdx3Cv (B,E,H,K), and α-syntrophin−/− mice (C,F,I,L). A–C: In cortex of wild-type mice strong AQP4 labeling was observed around vessels (A), whereas in mdx3Cv (B) and α-syntrophin−/− mice (C) the perivascular labeling was much weaker. A similar pattern was found in dentate gyrus (D–F) and cerebellum (G–I). J–L: In retina strong perivascular AQP4 labeling (arrowheads) is evident in all genotypes. Insets: Double labeling with the endothelial marker CD31 (blue) showed that the AQP4 immunofluorescence was peripheral to endothelial cells (arrows), corresponding to perivascular macroglial endfeet (arrowheads). GCL, granule cell layer; ILM, inner limiting membrane; ML, molecular layer; OPL, outer plexiform layer; PCL, Purkinje cell layer; asterix, pial surface. Scale bars: 50 μm, 5 μm (insets).
Figure 4
Figure 4
Electron micrographs showing subcellular distribution of AQP4 immunogold reactivity cortex (A–C) and retina (D–F) of wild-type (A,D), mdx3Cv (B,E) and α-syntrophin−/− mice (C,F). A,D: In cortex and retina of wild-type mice gold particles signaling AQP4 were much more abundant over macroglial endfoot membranes abutting capillary endothelial cells (End) and pericytes (P) than over macroglial membranes facing neurons (filled and open arrowheads denote the two membrane domains). A–C: In cortex of mdx3Cv and α-syntrophin−/− mice AQP4 immunogold reactivity over perivascular endfoot membranes was profoundly reduced compared to those of wild-types. D–F: In retina, however, AQP4 signaling gold particles were abundant over perivascular endfoot membranes of all genotypes. G: Quantitative analysis of AQP4 immunogold labeling over macroglial membranes in cortex and retina of wild-type, mdx3Cv and α-syntrophin−/− mice. The ordinate shows number of gold particles per μm of membrane quantified. The filled and open bars indicate labeling densities over endfoot membranes abutting capillaries (“Perivascular membranes”) and membranes facing neuropil, respectively (denoted with filled and open arrowheads in A–F, for easy comparison). SEM, number of profiles (N), and brackets with p values for comparison are indicated (cf. Materials and Methods). Scale bar, 0.5 μm.
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