ARTICLE |
Correspondence to: Hideho Ueda, Dept. of Neurosciences-NC30, the Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ß-Dystroglycan (ß-DG) is a dystrophin-associated glycoprotein that is expressed in skeletal muscle and other tissues. In the retina, dystrophin is present in the outer plexiform layer (OPL), where it is enriched under the photoreceptor cell membrane. In this study we determined the immunocytochemical localization of ß-DG at both light and electron microscopic levels. ß-DG immunoreactivity was detected at the inner limiting membrane, OPL, and around blood vessels. Immunoelectron microscopy detected ß-DG immunoreactive products under the photoreceptor cell membrane, which are the same regions of dystrophin localization. In addition, ß-DG was detected under the Müller cell membrane that is attached to the paravitreous or perivascular basement membrane. Our results suggest that ß-DG may interact with dystrophin in photoreceptor membranes. However, ß-DG-related interactions between Müller cells and basement membranes appear to be independent of dystrophin and raise the possibility that ß-DG interacts with other molecules. We speculate that ß-DG plays a role in maintaining the structural relationship between photoreceptor and bipolar cells or between Müller cells and basement membranes. (J Histochem Cytochem 46:185191, 1998)
Key Words: ß-dystroglycan, photoreceptor cell, Müller cell, immunoelectron microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dystrophin, a protein with a high molecular weight of about 420 kD, is absent in patients with Duchenne muscular dystrophy (DMD). This protein has been reported to be present under the sarcolemma of skeletal, cardiac, and smooth muscle fibers. In addition, dystrophin has been detected in the central nervous system, peripheral nerves, and retina (
Recent studies have indicated an oligomeric transmembrane complex in muscle cells that binds dystrophin and utrophin and interacts with several extracellular matrix proteins such as laminin, merosin, and agrin (-dystroglycan,
-DG), and one intracellular membrane-associated protein of the syntrophin family.
The human dystroglycan gene maps to chromosome 3p21 (- and ß-DG.
- and ß-DG are cleaved during or shortly after translation (
-DG interacts with laminin in the extracellular matrix (
It is generally accepted that the retinas of individuals with DMD are functionally and morphologically normal. However, recent studies have described abnormal electroretinograms (ERGs) in individuals with DMD, indicating a reduced amplitude of the b-wave under conditions of dark adaptation (- and ß-DG in the retina of normal and dystrophin-deficient mice (mdx mice), and their distributions were similar to those of dystrophin and utrophin.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and Tissue Preparations
Three adult male Wistar rats were used in the present study. The animals were anesthetized with ethyl ether and sodium pentobarbital and then perfused with 2% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.3. Their eyeballs were immediately enucleated and divided into anterior and posterior hemispheres. The retinas were gently removed and immersed in the same fixative for 60 min. After treatment with 30% sucrose in PBS, they were embedded in OCT compound and frozen at -80C.
Antibody
An anti-ß-DG antibody (NCL-43DAG) was purchased from Novocastra (Tyne, UK). This antibody recognizes 15 of the last 16 amino acids at the C-terminus of the human dystroglycan sequences (PKNMTPYRSPPPYVP-PCOOH).
Immunoblotting
Immunoblotting of ß-DG was performed as described previously (
Immunofluorescence Microscopy
Immunocytochemical procedures were performed as described previously (
Conventional Electron Microscopy
Some retinas were routinely fixed with 2.5% glutaraldehyde in PB for 60 min and 1% osmium tetroxide in PB for 60 min. After being rinsed in PBS, they were dehydrated in a graded series of ethanol concentrations and embedded in Quetol-812 (Nissin EM; Tokyo, Japan). Ultrathin sections were prepared, counterstained with uranyl acetate and lead citrate, and observed with a Hitachi H-600 electron microscope (Tokyo, Japan).
Immunoelectron Microscopy
The immunoperoxidase technique was used for immunoelectron microscopy. The 610µm cryosections, which had been immunostained with anti-ß-DG antibody, were subsequently incubated with rabbit anti-mouse IgG antibody conjugated to biotin and then with horseradish peroxidase conjugated to streptavidin (Nichirei; Tokyo, Japan). After being rinsed in PBS, they were fixed again by 0.25% glutaraldehyde in PB for 10 min. Then they were rendered visible by metal-enhanced DAB (Pierce; Rockford, IL) and treated with 1% osmium tetroxide in PB for 30 min. After routine dehydration in a graded series of ethanol and acetone concentrations, they were embedded in epoxy resin by the inverted gelatin capsule method. Finally, ultrathin sections were counterstained with uranyl acetate and lead citrate and observed in an electron microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoblotting Analysis
To check the specificity of the anti-ß-DG antibody, immunoblotting was performed with skeletal muscle supernatants (SM), retina supernatants (RS), and retina pellets (RP). Figure 1 shows immunoblotting of rat skeletal muscle and retina extracts with anti-ß-DG antibody. All lanes clearly show a 43-kD molecular weight band (arrowhead), which was most intense in the RP lane. Additional bands were also observed in all lanes. A 114-kD band was labeled intensely in the RS and RP lanes but was only faintly detectable in the SM lane. A 65-kD band was observed in all lanes, and a 29-kD band was observed only in the RP lane.
|
Confocal Laser Scanning Microscopic Analysis
ß-DG distribution in the retina was examined with immunofluorescence staining and observed by confocal laser scanning microscopy. ß-DG was detected in the inner limiting membrane (ILM) and around blood vessels of rat retinas. In addition, it was detected in the OPL like distinctive foci (Figure 2A), as well as dystrophin (
|
Electron Microscopic Analysis
ß-DG localization in the retina was investigated at the ultrastructural level. The ILM of the retina consists of paravitreous basement membranes and Müller cell membranes, and electron-dense regions are present under the Müller cell membranes (Figure 3, inset). ß-DG-immunoreactive products were discontinuously detected under the Müller cell membrane (Figure 3), and their localization was consistent with the electron-dense regions. Müller cells always encircled the blood vessels in the retina and attached to the basement membrane around the endothelial cells or vascular smooth muscle fibers (Figure 4A). They had electron-dense regions beneath the cell membrane as well as paravitreous ones. ß-DG immunoreactive products were detected under the Müller cell membrane that is attached to the perivascular basement membrane (Figure 4B) but was not detected beneath the sarcolemma of the vascular smooth muscle fibers (data not shown).
|
|
ß-DG localization in the rod spherule was compared with conventional sections (Figure 5). Two bipolar cell processes were separated by a rod spherule projection (Figure 5A). It was noted that electron-dense regions were present under the rod cell membrane facing bipolar cells but not facing horizontal cells. In a corresponding immunoelectron micrograph, ß-DG immunoreactive products were detected around two bipolar cell processes (Figure 5B) but not around horizontal cell processes or synaptic ridges (Figure 5C). ß-DG localization is consistent with the submembranous electron-dense regions, and its distribution is the same to dystrophin (Ueda et al., in press). The synaptic ribbon was not different from that of control sections, even though it had high electron density. Accordingly, it is impossible to state that ß-DG is expressed at synaptic ribbons.
|
Cone cells have two synapses, an invaginated synapse and a flat synapse (Figure 6). Invaginated synapses have horizontal cell processes and synaptic ribbons, but flat synapses have neither. Cone cell membranes facing the bipolar cell processes showed high electron density at both synapses as well as at the rod cell membrane. The inset to Figure 6 shows ß-DG immunoreactivity at a flat synapse under the cone cell membrane.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, ß-DG was detected in the rat retina using immunoblotting and immunocytochemistry. Immunofluorescence stainings showed that ß-DG was expressed in the INL, OPL, and around blood vessels. At the ultrastructural level, ß-DG was localized under Müller cell membranes that are attached to basement membranes, and under photoreceptor cell membranes facing bipolar processes. Considering these locations, ß-DG may function in a number of membranemembrane or membraneextracellular matrix interactions.
A monoclonal anti-ß-DG antibody showed some immunoreactive bands in addition to a 43-kD molecular weight band in the rat retinal extracts. It is assumed that ß-DG interacts with -DG and dystrophin/utrophin to connect cell membranes to the extracellular matrix in skeletal muscles. Therefore, larger molecular weight bands might indicate complexes of ß-DG and other proteins, and a small molecular weight band in the retinal pellet might indicate a novel ß-DG isoform. However, it is not known whether the ß-DG immunoreactive products represent ß-DG tightly associated with other proteins or spliced novel isoforms encoded by the same gene.
In this study, ß-DG was clearly detected under Müller cell membranes that were attached to paravitreous or perivascular basement membranes. Müller cells, the main glial cells in the vertebrate retina, penetrate all retinal layers perpendicularly. The plasma membrane of Müller cells is characterized by orthogonal arrays of particles (OAPs) that are revealed by the freeze-fracture replica method (
In our previous study, dystrophin was detected in rod and cone cells (
Recent studies have suggested that dystroglycan might function as a receptor for basement membrane components during epithelial morphogenesis (
In summary, ß-DG is localized under the Müller cell membrane attached to the paravitreous or perivascular basement membrane. In addition, it is localized at submembranous dense regions in the photoreceptor cells, along with dystrophin. However, further studies are needed to determine the function of ß-DG in the retina.
![]() |
Acknowledgments |
---|
We thank Dr Bruce D. Trapp (Department of Neurosciences, The Cleveland Clinic Foundation) for checking our manuscript. We also thank Ms Y. Kato and Ms K. Ariizumi for excellent assistance.
Received for publication May 19, 1997; accepted August 22, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahn AH, Kunkel LM (1993) The structure and functional diversity of dystrophin. Nature Genet 3:283-291[Medline]
Bowe MA, Deyst KA, Leszyc JD, Fallon JR (1994) Identification and purification of an agrin receptor from torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans. Neuron 12:1173-1180[Medline]
Campanelli JT, Roberds SL, Campbell KP, Scheller RH (1994) A role for dystrophin-associated glycoproteins and utrophin in agrin induced AChR clustering. Cell 77:663-674[Medline]
Drenckhahn D, Holbach M, Ness W, Schmitz F, Anderson LVB (1996) Dystrophin and the dystrophin-associated glycoprotein, ß-dystroglycan, co-localize in photoreceptor synaptic complexes of the human retina. Neuroscience 73:605-612[Medline]
Durbeej M, Larsson E, IbraghimovBeskrovnaya O, Roberds SL, Campbell KP, Ekblom P (1995) Non-muscle -dystroglycan is involved in epithelial development. J Cell Biol 130:79-91[Abstract]
Ervasti JM, Campbell KP (1993) A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol 122:809-823[Abstract]
Gee SH, Blacher RW, Douville PJ, Provost PR, Yurchenco PD, Carbonetto S (1993) Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin. J Biol Chem 268:14972-14980
Gee SH, Montanaro F, Lindenbaum MH, Carbonetto S (1994) Dystroglycan-alpha, a dystrophin associated glycoprotein, is a functional agrin receptor. Cell 77:675-686[Medline]
IbraghimovBeskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP (1992) Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 355:696-702[Medline]
IbraghimovBeskrovnaya O, Milatovich A, Ozcelik T, Yang B, Koepnick K, Francke U, Campbell KP (1993) Human dystroglycan; skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosomal localization. Hum Mol Genet 2:1651-1657[Abstract]
Landis DMD, Reese TS (1981) Membrane structure in mammalian astrocytes: a review of freeze-fracture studies on adult, developing, reactive and cultured astrocytes. J Exp Biol 95:35-48[Abstract]
Lenk U, Oexle K, Voit T, Ancker U, Hellner K-A, Speer A, Hübner C (1996) A cysteine 3340 substitution in the dystroglycan-binding domain of dystrophin associated with Duchenne muscular dystrophy, mental retardation and absence of the ERG b-wave. Hum Mol Genet 5:973-975
Linberg KA, Fisher SK (1988) Ultrastructural evidence that horizontal cell axon terminals are presynaptic in the human retina. J Comp Neurol 268:281-297[Medline]
Matsumura K, Tomé FMS, Collin H, Azibi K, Chaouch M, Kaplan J-C, Fardeau M, Campbell KP (1992) Deficiency of the 50k dystrophin-associated glycoprotein in severe childhood autosomal recessive muscular dystrophy. Nature 359:320-322[Medline]
Montanaro F, Carbonetto S, Campbell KP, Lindenbaum M (1995) Dystroglycan expression in the wild type and mdx mouse neu-ral retina: synaptic colocalization with dystrophin, dystrophin-related protein but not laminin. J Neurosci Res 42:528-538[Medline]
Newman E, Reichenbach A (1996) The Müller cell: a functional element of the retina. Trends Neurosci 19:307-312[Medline]
Ohlendieck K (1996) Towards an understanding of the dystrophin-glycoprotein complex: linkage between the extracellular matrix and the membrane cytoskeleton in muscle fibers. Eur J Cell Biol 69:1-10[Medline]
Ozawa E, Yoshida M, Suzuki A, Mizuno Y, Hagiwara Y, Noguchi S (1995) Dystrophin-associated proteins in muscular dystrophy. Hum Mol Genet 4:1711-1716[Abstract]
Pillers DM, Bulman DE, Weleber RG, Sigesumund DA, Musarella MA, Powell BR, Murphey WH, Westall C, Panton C, Becker LE, Worton RG, Ray PN (1993) Dystrophin expression in the human retina is required for normal function as defined by electroretinography. Nature Genet 4:82-86[Medline]
Schmitz F, Holbach M, Drenckhahn D (1993) Colocalization of retinal dystrophin and actin in postsynaptic dendrites of rod and cone photoreceptor synapses. Histochemistry 100:473-479[Medline]
Sigesmund DA, Weleber RG, Pillers DM, Westall CA, Panton CM, Powell BR, Héon E, Murphey WH, Musarella MA, Ray PN (1994) Characterization of the ocular phenotype of Duchenne and Becker muscular dystrophy. Ophthalmology 101:856-865[Medline]
Suzuki A, Yoshida M, Hayashi K, Mizuno Y, Hagiwara Y, Ozawa E (1994) Molecular organization at the glycoprotein-complex-binding site of dystrophin. Three dystrophin-associated proteins bind directly to the carboxy-terminal portion of dystrophin. Eur J Biochem 220:289-292
Suzuki A, Yoshida M, Ozawa E (1995) Mammalian 1- and ß1-syntrophin bind to the alternative splice-prone region of the dystrophin COOH terminus. J Cell Biol 128:373-381[Abstract]
Suzuki A, Yoshida M, Yamamoto H, Ozawa E (1992) Glycoprotein-binding site of dystrophin is confined to the cystein-rich domain and the first half of the carboxy-terminal domain. FEBS Lett 308:154-160[Medline]
Ueda H, Baba T, Terada N, Kato Y, Tsukahara S, Ohno S Dystrophin in rod spherules; submembranous dense regions facing bipolar cell processes. Histochemistry, in press
Ueda H, Kato Y, Baba T, Terada N, Fujii Y, Tsukahara S, Ohno S (1997) Immunocytochemical study of dystrophin localization in cone cells of mouse retinas. Invest Ophthalmol Vis Sci 38:1627-1630[Abstract]
Ueda H, Kobayashi T, Mitsui K, Tsurugi K, Tsukahara S, Ohno S (1995) Dystrophin localization at presynapse in rat retina revealed by immunoelectron microscopy. Invest Ophthalmol Vis Sci 36:2318-2322[Abstract]
Wolburg H, Berg K (1988) Distribution of orthogonal arrays of particles in the Müller cell membrane of the mouse retina. Glia 1:246-252[Medline]
Yamada H, Shimizu T, Tanaka T, Campbell KP, Matsumura K (1994) Dystroglycan is a binding protein of laminin and merosin in peripheral nerve. FEBS Lett 352:49-53[Medline]
Yotsumoto S, Fujiwara H, Horton JH, Mosby TA, Wang X, Cui Y, Ko MSH (1996) Cloning and expression analyses of mouse dystroglycan gene: specific expression in maternal decidua at the peri-implantation stage. Hum Mol Genet 5:1259-1267[Medline]