Brain dystrophin-glycoprotein complex: Persistent expression of beta-dystroglycan, impaired oligomerization of Dp71 and up-regulation of utrophins in animal models of muscular dystrophy

Abstract

Aside from muscle, brain is also a major expression site for dystrophin, the protein whose abnormal expression is responsible for Duchenne muscular dystrophy. Cognitive impairments are frequently associated with this genetic disease, we therefore studied the fate of brain and skeletal muscle dystrophins and dystroglycans in dystrophic animal models. brain predominantly as a monomer. This suggests an association of β-dystroglycan with membranes at the vascular-glial interface in the forebrain. In contrast to dystrophic skeletal muscle fibres, dystrophin deficiency does not trigger a reduction of all dystroglycans in the brain, and utrophins may partially compensate for the lack of brain dystrophins. Abnormal oligomerization of the dystrophin isoform Dp71 might be involved in the pathophysiological mechanisms underlying abnormal brain functions.

Background

Aside from muscle, brain is also a major expression site for dystrophin, the protein whose abnormal expression is responsible for Duchenne muscular dystrophy. Cognitive impairments are frequently associated with this genetic disease, we therefore studied the fate of brain and skeletal muscle dystrophins and dystroglycans in dystrophic animal models.

Results

brain predominantly as a monomer.

Conclusions

This suggests an association of β-dystroglycan with membranes at the vascular-glial interface in the forebrain. In contrast to dystrophic skeletal muscle fibres, dystrophin deficiency does not trigger a reduction of all dystroglycans in the brain, and utrophins may partially compensate for the lack of brain dystrophins. Abnormal oligomerization of the dystrophin isoform Dp71 might be involved in the pathophysiological mechanisms underlying abnormal brain functions.

Background

].

]. One factor which probably makes pathophysiological studies of the dystrophic central nervous system more difficult is the greater complexity of dystrophin and utrophin isoforms present in the brain.

]. Our analysis of these mutant strains indicates that β-dystroglycan appears to be located at the endothelial-glial interface in the forebrain and that not all dystroglycans are reduced in dystrophic brain, making it different from dystrophic muscle fibres. Possibly an impaired oligomerization of the major brain Dp71 isoform plays a role in the molecular pathogenesis in the dystrophic central nervous system.

Results

).

-3cv mice.

, both normal muscle and fibres from both dystrophic animal models exhibit almost exclusively peripheral staining for spectrin establishing the integrity of the cryosections analysed.

-3cv mice (c, f, i, l, q, r, u, x). Bar = 60 μm.

.

Shown are cryosections indirectly labeled with rhodamine-conjugated antibodies to the neurofilament of apparent 68 kDa (a), the glial fibrillary acidic protein (b, c) and von Willebrand factor (d). Sections (a) to (d) were indirectly double-labeled with a fluorescein-conjugated antibody against β-dystroglycan. To demonstrate the specificity of the antibody to von Willebrand factor, rat aorta sections are shown in (e) (Haematoxylin & Eosin staining) and (f) (immunofluorescence labeled). In (a), bar = 20 μm; in (b) and (d), bar = 40 μm; in (c), bar = 10 μm; and in (e) and (f), bar = 60 μm.

).

-3cv mice (c, f, i, l, o). Bar = 40 μm.

). Thus, the decrease in dystrophin-associated glycoproteins in skeletal muscle is a specific resu lt of the deficiency of dystrophin, and not a consequence of general muscle cell destruction in dystrophic fibres.

-3cv fibres, respectively. The position of immuno-decorated protein bands is indicated by arrow heads.

).

) is indicated on the left.

-3cv brain, respectively. The position of immuno-decorated protein bands is indicated by arrow heads.

). This strongly suggests that the decrease in the relative electrophoretic mobility of Dp71 in normal brain microsomes is a specific result of crosslinker-induced stabilization of native membrane complexes.

) is indicated on the left.

Discussion

].

]. Although β-dystroglycan expression is preserved, this integral membrane protein might not be properly positioned in order to anchor extracellular α-dystroglycan to the outside of the membrane. Compensatory mechanisms to counteract the loss of dystrophin isoforms may induce conformational changes in β-dystroglycan units that interfere with stablising interactions within dystroglycan sub-complexes. Therefore, the preservation of β-dystroglycan does not seem to rescue the extracellular dystroglycan form.

brain. Although Dp71 co-localizes with β-dystroglycan, the lack of full-length brain dystrophin seems to trigger a disturbed organization of the dystroglycan sub-complex resulting in a drastic reduction in the extracellular dystroglycan isoform. These findings show that we still have an incomplete understanding of the individual functions of dystrophin isoforms and of the interaction between short and long dystrophins in different tissues.

]. Thus, probably a combination of different primary genetic defects in the DMD gene and variations in compensatory mechanisms result in the different degrees of mental insufficiencies in dystrophic children.

Conclusions

brain as a monomeric protein. Thus, the lack in brain dystrophins does not necessarily lead to a loss in all associated glycoproteins and possibly abnormal oligomerization of the brain dystrophin might play a role in the molecular pathogenesis of abnormal brain functions in muscular dystrophy.

Materials

Fluorescein-, rhodamine- or peroxidase-conjugated secondary antibodies were purchased from Boehringer Mannheim (Lewis, East Sussex, UK). Commercially available primary antibodies were from Novocastra Laboratories Ltd. (Newcastle upon Tyne, UK), Upstate Biotechnology (Lake Placid, NY, USA) and Sigma Chemical Company (Poole, Dorset, UK), and Texas Red-labeled α-bungarotoxin was purchased from Molecular Probes Europe BV (Leiden, The Netherlands). Superfrost Plus positively-charged microscope slides were from Menzel Glaesser (Braunschweig, Germany). Fuji Neopan 400ASA B/W photographic film was obtained from Fuji Photo Film Co. (Tokyo, Japan) and Kodacolor Gold 400ASA VR film from Eastman Kodak Company (Rochester, NY). Protease inhibitors and acrylamide were purchased from Boehringer Mannheim (Lewis, East Sussex, UK). Peroxidase-conjugated lectins were purchased from EY Labs (San Mateo, CA, USA). Western blotting chemiluminescence substrates and chemical crosslinkers were obtained from Pierce & Warriner (Chester, Cheshire, UK). Immobilon-P nitrocellulose was from Millipore Corporation (Bedford, MA, USA). All other chemicals were of analytical grade and purchased from Sigma Chemical Company (Poole, Dorset, UK).

Antibodies

]. The peptide had been synthesized and coupled to KLH carrier by Research Genetics (Huntington, AL).

Animal models

muscle and the forebrain region, quick-frozen in liquid nitrogen-cooled isopentane, transported on dry ice and stored at -70°C prior to cryosectioning. For immunoblot analysis, total brain and bulk skeletal muscle were dissected, quick-frozen in liquid nitrogen, transported in a container with dry ice and then stored at -70°C prior to homogenization.

Immunofluorescence microscopy

]. Photographs were taken on Fuji Neopan 400ASA B/W photographic film or Kodak Gold Kodacolor 400ASA VR film. For double-staining procedures, a mixture of the appropriate primary antibodies were applied to tissue sections for 1 h at 37°C, cryosections washed, and then separately incubated for 30 min each with the appropriate secondary antibodies. In case of antibodies which had been generated in the same animal species, photographic images were obtained from concurrent areas in serial sections, and the labeling results overlayed.

Isolation of muscle and brain membranes

] using bovine serum albumin as a standard.

Chemical crosslinking analysis

] was added and the solution incubated for 15 min at 37°C before being subjected to electrophoretic separation.

Gel electrophoresis, lectin staining and immunoblotting

]. Densitometric scanning of enhanced chemiluminescence blots was performed on a Molecular Dynamics 300S computing densitometer (Sunnyvale, CA) with ImageQuant V3.0 software.

Acknowledgements

Research was supported by project grants from the Irish Health Research Board (HRB-01/98) and Enterprise Ireland, Dublin (SC/2000/386), and a European travel grant from the Royal Society, London and the Royal Irish Academy, Dublin. The authors would like to thank Drs. H. Jockusch (University of Bielefeld, Germany), K.P. Campbell (University of Iowa, IA, USA) and S. Winder (University of Glasgow, Scotland) for providing our lab with animal models and antibodies.