Brain dystrophin-glycoprotein complex: Persistent expression of β-dystroglycan, impaired oligomerization of Dp71 and up-regulation of utrophins in animal models of muscular dystrophy
© Culligan et al; licensee BioMed Central Ltd. 2001
Received: 29 November 2000
Accepted: 2 February 2001
Published: 2 February 2001
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.
All dystrophin-associated glycoproteins investigated were reduced in dystrophic muscle fibres. In Dp427-deficient mdx brain and Dp71-deficient mdx-3cv brain, the expression of α-dystroglycan and laminin was reduced, utrophin isoforms were up-regulated and β-dystroglycan was not affected. Immunofluorescence localization of β-dystroglycan in comparison with glial, endothelial and neuronal cell markers revealed co-localization of von Willebrand factor with β-dystroglycan. Its expression at the endothelial-glial interface was preserved in dystrophin isoform-deficient brain from mdx and mdx-3cv mice. In addition, chemical crosslinking revealed that the Dp71 isoform exists in mdx 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.
The main hypotheses of how deficiency in dystrophin triggers muscular dystrophy suggest that the lack of this membrane cytoskeletal component weakens the sarcolemmal integrity, causes abnormal Ca2+-homeostasis and/or impairs proper clustering of ion channel complexes [1, 2]. Extensive biochemical and cell biological studies have demonstrated that one of the major functions of muscle dystrophin is to act as an actin-binding protein which mediates a link between the extracellular matrix component laminin and the sub-sarcolemmal membrane cytoskeleton [3,4]. Integral or surface-associated proteins that are relatively tightly connected with dystrophin are represented by α-,β-, γ-, and δ-sarcoglycan , α- and β-dystroglycan , sarcospan , α-, β1-, and β2-syntrophin , α- and β-dystrobrevin , laminin-2  and cortical actin . The backbone of this sarcolemma-spanning protein assembly is formed by the dystroglycans . The extreme carboxy-terminus of 43 kDa β-dystroglycan contains a binding site for the second half of the hinge-4 region and the cysteine-rich domain of Dp427 , thereby indirectly connecti ng the actin membrane cytoskeleton via the amino-terminus of the dystrophin molecule to the surface membrane . Since β-dystroglycan is also tightly associated with the peripheral merosin-binding protein α-dystroglycan, this complex provides a stable linkage to the laminin α2-chain in the basal lamina .
Deficiency in dystrophin triggers the disintegration of complexes normally formed by the above listed sarcolemmal components and thereby renders muscle fibres from patients afflicted with Duchenne muscular dystrophy (DMD) more susceptible to necrosis [1, 3]. In analogy to the pathobiochemical findings in DMD [3, 14], the dystrophic animal model mdx mouse also exhibits a drastic reduction in all dystrophin-associated glycoproteins in bulk skeletal muscle [15, 16]. This might explain at least partially the decreased osmotic stability  and higher vulnerability of stretch-induced injury  in dystrophin-deficient muscle fibres. An abnormal increase in cytosolic Ca2+- levels might trigger a drastic inc rease in net protein degradation and might be one of the initial steps in the molecular pathogenesis of inherited muscular dystrophy [19,20,21]. That the other members of the dystrophin -glycoprotein complex, besides dystrophin, play a role in the DMD pathology, is demonstrated by the fact that primary abnormalities in sarcoglycans and laminin are responsible for certain forms of limb-girdle muscular dystrophy and congenital muscular dystrophy, respectively [5, 22]. In contrast to muscle, much less is known about the molecular mechanisms underlying brain abnormalities in the most frequent neuromuscular disease in humans [23, 24]. 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.
Seven promoters drive the tissue-specific expression of various dystrophin protein (Dp) isoforms from the human DMD gene , i.e. Dp427-M in skeletal and cardiac muscle, Dp427-B in brain, Dp427-P in Purkinje neurons, Dp-260 R in retina, Dp -140 - B/K in brain and kidney, Dp -116-S in Schwann cells, Dp-71-B/U in brain and many non-muscle tissues . In addition, dystrophin-related proteins are represented by brain DRP-2  and the autosomally-encoded dystrophin homologue utrophin, which forms a full-length 395 kDa isoform (Up395)  and two truncated molecular species named Up116 and Up71, also referred to as G-and U-utrophin . Besides full-length brain Dp427 and a relatively low-abundance, carboxy-terminal isoform termed brain Dp140, in the central nervous system the major dystrophin isoform is represented by Dp71 . While Dp427 was shown to be present in cortical neurons, hippocampal neurons and cerebellar Purkinje cells , probably mostly associated in these cell types with the postsynaptic density , the two smaller dystrophin brain isoforms were described to be associated with microvasular glial cells . A developmental study suggests that dystrophin expression in perivascular astrocytes coincides with the formation of the blood-brain barrier . Dystroglycans are also present in brain [33, 34] and a subpopulation localizes to the glial-vascular interface . Recently, Blake et al.  showed that different dystrobrevin isoforms are present in neuronal versus glial dystrophin complexes. With respect to dystrophin-related proteins, full-length utrophin is more widely distributed in the central nervous system  and is possibly involved in the maintenance of regional specialization of the brain . To complement these neurobiological studies and in order to determine the fate of dystroglycans in dystrophin-deficient forebrain, we employed two established genetic animal models. The mdx mouse is missing Dp427 due to a point mutation in exon 23 , while a mutation in exon 65 in the mdx-3cv mouse affects the splicing of both the 4.8 and 14 kb dystrophin mRNAs resulting in the additional loss of the Dp71 isoform . Neurobehavioral studies have shown that the dystrophic animal models used in this study exhibit moderate alterations in associative learning and deficits in long-term consolidation memory [24, 40,41,42]. 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.
Although the X-linked inherited disorder Duchenne muscular dystrophy (DMD) is primarily considered a muscle disease and most patients die of respiratory or cardiac failure , in a subpopulation of affected children non-progressive mental retardation preceeds degeneration of the muscular system . These mental abnormalities do not correlate with the stage of the muscle disease  and can not be attributed to abnormal motor development . Since all DMD patients experience a decrease in strength of limb and torso muscles, but only approximately one-third of dystrophic children suffer from cognitive impairments, it is believed that differences exist in the pathophysiolgical mechanisms between the central nervous system and muscle tissues [23, 24]. DMD children accomplish performance tasks at a normal level, but their verbal intelligence quotient is significantly lower as compared to age-matched normal boys . Possibly cerebral or cerebellar hypermetabolism is involved in cognitive impairments in certain DMD patients , but no consistent abnormalities are detectable in dystrophic brain tissues .
Based on this lack of understanding of the exact neurobiology of DMD, we have performed here a comparative analysis of the expression of dystrophins and dystroglycans in brain and muscle tissues from animal models of muscular dystrophy. Forebrain β-dystroglycan was clearly shown to co-localize with the endothelial marker von Willebrand factor and it is not drastically affected in its relative abundance in brain lacking all neuronal dystrophin isoforms. The localization of this relatively abundant glycoprotein at the endothelial-glial interface agrees with previous immunolocalization studies on dystrophin-associated proteins [31, 32, 51,52,53,54]. Dystrophin isoforms of varying length, dystrobrevin and β-dystroglycan appear to be enriched around blood vessels in astrocytic endfeet in the cerebellum and at blood-ocular barrier sites in the retina [51,52,53,54]. Here we can show that the cellular localization of this integral membrane component at the endothelial-glial interface is neither changed in Dp427-deficient mdx forebrain or in Dp71-deficient mdx-3cv forebrain. Thus, in contrast to dystrophic mdx and DMD skeletal muscle fibres, which show a greatly reduced expression of sarcolemmal β-dystroglycan [3, 15, 16], this usually dystrophin-associated glycoprotein experiences a different fate during pathophysiological changes in the central nervous system of dystrophic mice. However, the relative expression of α-dystroglycan is reduced in dystrophic brain. This is unexpected, since both α- and β-dystroglycan are produced by post-translational cleavage of the product of a single transcript . 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.
Possibly up-regulation of utrophin isoforms partially compensates for the lack of brain dystrophins and thereby helps anchoring β-dystroglycans. This idea agrees with previous studies of extraocular muscle fibres from mdx and mdx-utrn-/- mice [55, 56]. In contrast to the neuromuscular junction-specific localization of utrophin in normal skeletal muscle , in dystrophin-deficient mdx extraocular muscle the full-length isoform of utrophin of apparent 395 kDa is up-regulated in its relative expression and also found in n on-junctional regions of the sarcolemma . This replacement of dystrophin Dp427 by the large utrophin isoform seems to spare a large proportion of the extraocular muscle population from degeneration. However, mdx-utrn-/- mice lacking both dystrophin and utrophin exhibit severe dystrophic changes in these muscle groups strongly suggesting that the endogenous up-regulation of utrophin protects extraocular muscle in dystrophinopathies . A similar protective mechanism might occur in dystrophic brain regions. We could previously show that most members of the dystrophin super-family of proteins, which share the carboxy-terminal binding domain for β-dystroglycan, exhibit very comparable biochemical properties [59, 60]. This was also confirmed for brain isoforms of dystrophin . Since brain utrophins co-localize with the dystroglycan sub-complex in the forebrain, it seems likely that an up-regulation of utrophins anchors these components in Dp427- or Dp71-deficient membranes. Dp71 alone does not appear to properly oligomerize and anchor dystroglycans in mdx 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.
In contrast to established changes in the expression of dystrophins and utrophins in dystrophic brain [23,30,62], relatively little is known about the fate of dystrophin/utrophin-associated glycoproteins in human DMD brain. In contrast to mdx brain, DMD patient specimens appear to exhibit a reduction in β-dystroglycan levels [16, 63]. However, representative surveys of large patient populations with a varying degree of mental retardation have not yet been performed making it difficult to compare findings from genetic animal models with patient data. In this respect, the finding presented in this study that the major brain dystrophin isoform Dp71 does not appear to properly oligomerize in mdx brain might also be relevant for the human disease condition. The lack of crosslinker-induced complex stabilization indicates that Dp71 might trigger abnormal anchoring of dystroglycans, although it is present at normal concentrations. This in turn might destabilize certain brain structures and/or signal transduction pathways normally relying on the integrity of brain dystrophin-glycoprotein complexes. Since Ca2+-levels were found to be abnormal in dystrophic brain , similar pathophysiological changes, as suggested to be involved in muscular degeneration [19,20,21], could also render certain brain cells more susceptible to necrosis. An increased influx of Ca2+-ions might trigger cell destruction not only in Dp71-deficient cells but also in cellular structures with Dp71 molecules not capable of properly forming complexes with β-dystroglycan. In the dystrophic forebrain, abnormal anchoring of dystroglycans might therefore affect the proper establishment of the blood-brain barrier. However, since the cognitive impairment in DMD is non-progressive and exhibits great variations between individual patients, only a sub-population of brain cells may be affected by this pathophysiological mechanism. Deletions in the exon 45-52 region of the DMD gene have been reported to be associated with an increased incidence of cognitive abnormalities . In these patients only the expression of the Dp 427 and Dp140 isoforms is impaired, but not the Dp71 protein . 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.
In conclusion, this report demonstrates that β-dystroglycan is not present at high concentrations in central neurons of the forebrain region, but seems to be mostly located at the interface between endothelial cells and glia. These structures possibly represent endfeet on astrocytes at the blood-brain barrier. In dystrophic forebrain, β-dystroglycan expression is not drastically affected, possibly due to the up-regulation of utrophin isoforms which partially compensate for the deficiency in brain dystrophins. Chemical crosslinking analysis showed that Dp71 exists in contrast to its normally oligomeric form in mdx 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 and methods
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).
Monoclonal and polyclonal antibodies employed in this study were characterized as previously described [66, 67]. Monoclonal antibodies NCL-43 against β-dystroglycan, NCL-a-SARC against α-sarcoglycan, DYS-1 to the Dp427 rod-domain, DYS-2 to the Dp427 carboxy-terminus, NCL-DRP1 to the carboxy -terminus of full-length utrophin and NCL-SPEC2 against spectrin were from Novocastra Laboratories Ltd. (Newcastle upon Tyne, UK). Monoclonal antibodies VIA41 to α-dystroglycan and c464.6 to the α-subunit of the Na+/K+-ATPase were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Polyclonal antibodies to von Willebrand factor, laminin and the glial fibrillary acidic protein, as well as monoclonal antibody NR4 to the neurofilament of apparent 68 kDa were obtained from Sigma Chemical Company (Poole, Dorset, UK). A polyclonal antibody which recognizes the carboxy-terminal domain of the utrophin isoforms Up395, Up116 and Up71  was a generous gift of Dr. Steve Winder (University of Glasgow). Monoclonal antibody IIID5 against the α1-subunit of the dihydropyridine receptor was a generous gift of Dr. Kevin P. Campbell (University of Iowa, Iowa City, IA). An antibody to the extreme carboxy-terminus of α-sarcoglycan was raised by 4 monthly injections of a peptide representing the last 15 residues of the carboxy-terminus  using a standard immunization protocol . The peptide had been synthesized and coupled to KLH carrier by Research Genetics (Huntington, AL).
Muscle and brain samples from the mdx mouse, which lacks the Dp427 isoform of dystrophin due to a point mutation in exon 23 , and from the mdx-3cv mouse, which has a mutation in exon 65 that affects the splicing of both the 4.8 and 14 kb dystrophin mRNAs causing a loss of all dystrophin isoforms including the major brain dystrophin isoform Dp71 , were a generous gift from Dr. Harald Jockusch (Department of Developmental Biology, University of Bielefeld, Germany). For immunofluorescence microscopy, tissue specimens were taken from the tibialis anterior 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.
For immunolabeling of muscle and brain tissue sections, 12 μm cryosections were prepared using a standard cryostat (Microm, Heidelberg, Germany) and mounted on Superfrost Plus positively-charged microscope slides. Fixation, blocking, incubation with primary antibodies, washing steps, incubation with secondary antibodies, as well as photography was performed by established methodology . 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
In order to compare the relative expression levels of members of the dystrophin-glycoprotein complex by immunoblotting, established protocols for the isolation of microsomal membranes from skeletal muscle  and brain  were employed. To minimize proteolytic degradation of membrane proteins, all buffers contained a protease inhibitor cocktail (0.2 mM pefabloc, 1.4 μM pepstatin, 0.15 μM aprotinin, 0.3 μM E-64, 1 μM leupeptin, 0.5 μM soybean trypsin inhibitor, and 1 mM EDTA) and all procedures were performed in a cold room at 0-4°C. Membrane pellets were resuspended at a protein concentration of 10 mg/ml and used immediately for gel electrophoretic analysis or quick-frozen in liquid nitrogen and then stored at -70°C prior to further usage. Protein concentration was determined by the method of Bradford  using bovine serum albumin as a standard.
Chemical crosslinking analysis
Chemical crosslinking was performed as previously described in detail [45, 66]. Microsomes (1 mg protein) were diluted to a final volume of 500 μl with 50 mM HEPES, pH 8.0 at 25°C. Using a stock solution of 5 mg/ml chemical crosslinker, bis-sulfosuccinimidyl-suberate (BS3) was added to the membrane suspension at a final concentration of 200 μg cross-linker per mg membrane protein. Since the cross -linker BS3 is water-soluble, it was dissolved in 50 mM citrate buffer, pH 5.0 in order to retard hydrolysis. Samples were incubated for 30 min with constant agitation at 25°C and then the crosslinking reactions terminated by the addition of 50 μl of 1 M ammonium acetate per ml reaction mixture. An equal volume of reducing sample buffer  was added and the solution incubated for 15 min at 37°C before being subjected to electrophoretic separation.
Gel electrophoresis, lectin staining and immunoblotting
Gel electrophoretic separation using 5% or 7% (w/v) resolving gels with a 5% (w/v) stacking gel in the presence of sodium dodecyl sulfate and dithiotreitol was performed for 200 Vh employing a Mini-MP3 electrophoresis system from Bio-Rad Laboratories (Hempel Hempstead, Herts., UK), whereby 25 μg protein was loaded per well [66, 73]. Chemically crosslinked samples were separated on gels lacking a stacking gel system. Nitrocellulose replica of polyacrylamide gels were produced as described by Towbin et al. . Blot overlays with peroxidase-conjugated lectins (MPA, Maclura pomifera lectin; WGA, Tritium vulgaris lectin) were carried out as previously described . For immunolabeling, nitrocellulose sheets were blocked and incubated with primary and secondary antibodies as previously described . Immunodecoration was evaluated by the enhanced chemiluminescence technique . Densitometric scanning of enhanced chemiluminescence blots was performed on a Molecular Dynamics 300S computing densitometer (Sunnyvale, CA) with ImageQuant V3.0 software.
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.
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