The role of utrophin and Dp71 for assembly of different dystrophin-associated protein complexes (DPCs) in the choroid plexus and microvasculature of the brain

In the brain, utrophin is present in the choroid plexus epithelium and vascular endothelial cells, whereas the short C-terminal isoform of dystrophin (Dp71) is localized in the glial end-feet surrounding blood vessels. Both proteins serve as anchors for the so-called dystrophin-associated protein complex (DPC), composed of isoforms of syntrophin, dystroglycan and dystrobrevin. Numerous transporter proteins and channels have a polarized distribution in vascular endothelial cells and in glial end-feet, suggesting an association with the DPC. We investigated the composition and localization of the DPC in dependence on the anchoring proteins in mice lacking either utrophin (utrophin0/0) or dystrophin isoforms (mdx3Cv). Three distinct complexes were identified: (i) associated with utrophin in the basolateral membrane of the choroid plexus epithelium, (ii) associated with utrophin in vascular endothelial cells, and (iii) associated with Dp71 in the glial end-feet. Upon ablation of utrophin or Dp71, the corresponding DPCs were disrupted and no compensation of the missing protein by its homologue was observed. Association of the water channel aquaporin 4 with the glial DPC likewise was disrupted in mdx3Cv mice. These results demonstrate the essential role of utrophin and Dp71 for assembly of the DPC and suggest that these proteins contribute to the proper functioning of the cerebrospinal fluid and blood-brain barriers.     

A sea urchin gene encoding dystrophin-related proteins

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least six additional products: two non-muscle dystrophin isoforms transcribed from promoters located in the 5'-end region of the gene and four smaller proteins transcribed from internal promoters located further downstream. Several other genes, encoding evolutionarily related proteins, have been identified. These include a structurally very similar gene in vertebrates encoding utrophin (DRP1), which is closely related to dystrophin, and a number of small and simple genes in vertebrates or invertebrates encoding proteins similar to some of the small products of the DMD gene. We have isolated a sea urchin gene showing very strong sequence and structural homology with the DMD and utrophin genes. Sequence and intron/exon structure similarities suggest that this gene is related to a precursor of both the DMD gene and the gene encoding utrophin. The sea urchin gene has the unique complex structure of the DMD gene. There is at least one, and possibly more, product(s) transcribed from internal promoters, as well as a large product of >300 kDa containing at least three of the four major domains of dystrophin. The small product seems to be evolutionarily related to Dp116, one of the small products of the human DMD gene. Partial characterization of this gene helped us to construct an evolutionary tree connecting the vertebrate dystrophin gene family with related genes in invertebrates. The constructed evolutionary tree also implies that the vertebrate small and simple structured gene encoding a Dp71-like protein, called DRP2 , evolved from the dystrophin/utrophin ancestral large and complex gene by a duplication of only a small part of the gene.      

Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina

The abnormal retinal neurotransmission observed in Duchenne muscular dystrophy (DMD) patients and in some genotypes of mice lacking dystrophin has been attributed to altered expression of short products of the dystrophin gene. We have investigated the potential role of Dp71, the most abundant C-terminal dystrophin gene product, in retinal electrophysiology. Comparison of the scotopic electroretinograms (ERG) between Dp71-null mice and wild-type (wt) littermates revealed a normal ERG in Dp71-null mice with no significant changes of the b-wave amplitude and kinetics. Analysis of DMD gene products, utrophin and dystrophin-associated proteins (DAPs), showed that Dp71 and utrophin were localized around the blood vessels, in the ganglion cell layer (GCL), and the inner limiting membrane (ILM). Dp71 deficiency was accompanied by an increased level of utrophin and decreased level of beta-dystroglycan localized in the ILM, without any apparent effect on the other DAPs. Dp71 deficiency was also associated with an impaired clustering of two Müller glial cell proteins-the inwardly rectifying potassium channel Kir4.1 and the water pore aquaporin 4 (AQP4). Immunostaining of both proteins decreased around blood vessels and in the ILM of Dp71-null mice, suggesting that Dp71 plays a role in the clustering and/or stabilization of the two proteins. AQP4 and Kir4.1 may also be involved in the regulation of the ischemic process. We found that a transient ischemia resulted in a greater damage in the GCL of mice lacking Dp71 than in wt mice. This finding points at a crucial role played by Dp71 in retinal function.     

Altered blood-brain barrier development in dystrophic MDX mice

In order to ascertain whether the alterations of the blood-brain barrier (BBB) seen in adult dystrophic mdx-mice [Glia 42 (2003) 235], a human model of Duchenne muscular dystrophy (DMD), are developmentally established and correlated with other dystrophin isoforms which are localized at the glial-vascular interface, we used immunocytochemistry to investigate the expression of dystrophin isoforms (Dp71) during BBB development in mdx fetuses and in adult mice. Parallelly, we used Western blot, immunocytochemistry and immunogold electron microscopy to analyze the expression of the zonula occludens (ZO-1), aquaporin-4 (AQP4) and glial fibrillary acidic (GFAP) proteins as endothelial and glial markers, and we evaluated the integrity of the mdx BBB by means of intravascular injection of horseradish peroxidase (HRP). The results show reduced dystrophin isoforms (Dp71) in the mdx mouse compared with the control, starting from early embryonic life. Endothelial ZO-1 expression was reduced, and the tight junctions were altered and unlabeled. AQP4 and GFAP glial proteins in mdx mice also showed modifications in developmental expression, the glial vascular processes being only lightly AQP4- and GFAP-labeled compared with the controls. Confocal microscopy and HRP assays confirmed the alteration in vessel glial investment, GFAP perivascular endfoot reactivity being strongly reduced and BBB permeability increasing. These results demonstrate that a reduction in dystrophin isoforms (Dp71) at glial endfeet leads to an altered development of the BBB, whose no-closure might contribute to the neurological dysfunctions associated with DMD.     

Effect of beta-dystroglycan processing on utrophin/Dp116 anchorage in normal and mdx mouse Schwann cell membrane

In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and beta-dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the dystroglycan complex by cleaving the extracellular domain of beta-dystroglycan. Metalloproteinase creates a 30 kDa fragment of beta-dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the beta-dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa beta-dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of beta-dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa beta-dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of beta-dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.     

Utrophin and dystrophin-associated glycoproteins in normal and dystrophin deficient cardiac muscle

In this study, various members of the dystrophin family (dystrophin, the short dystrophin product Dp 71, utrophin and DRP2), and different members of the dystrophin-associated glycoprotein (DAG) complex (beta-dystroglycan, alpha-, beta-, gamma- and delta-sarcoglycans) were localized in bovine cardiac muscle using a battery of specific antibodies. We have established that dystrophin is exclusively associated with beta-dystroglycan and both alpha- and delta-sarcoglycans in cardiac muscle cell membranes. In contrast, utrophin is a specific component of intercalated disks together with beta- and gamma-sarcoglycans, while beta-dystroglycan, alpha- and delta-sarcoglycans are not present. Dp 71 is mainly localized at the T tubule transverse area. In dystrophin deficient cardiac muscle, utrophin and beta-sarcoglycan were observed in intercalated disks and at the sarcolemma of each cardiocyte. Our results revealed that complexes of associated glycoproteins differ in cardiac muscle when associated with dystrophin or utrophin. Despite the described sequence homologies between dystrophin and utrophin, the present results indicate that these proteins have different roles in some specific cardiac cell areas.     

Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure

The X-linked muscle wasting disease Duchenne muscular dystrophy is caused by the lack of dystrophin in muscle. Protein structure predictions, patient mutations, in vitro binding studies and transgenic and knockout mice suggest that dystrophin plays a mechanical role in skeletal muscle, linking the subsarcolemmal cytoskeleton with the extracellular matrix through its direct interaction with the dystrophin-associated protein complex (DAPC). Although a signaling role for dystrophin has been postulated, definitive data have been lacking. To identify potential non-mechanical roles of dystrophin, we tested the ability of various truncated dystrophin transgenes to prevent any of the skeletal muscle abnormalities associated with the double knockout mouse deficient for both dystrophin and the dystrophin-related protein utrophin. We show that restoration of the DAPC with Dp71 does not prevent the structural abnormalities of the post-synaptic membrane or the abnormal oxidative properties of utrophin/dystrophin-deficient muscle. In marked contrast, a dystrophin protein lacking the cysteine-rich domain, which is unable to prevent dystrophy in the mdx mouse, is able to ameliorate these abnormalities in utrophin/dystrophin-deficient mice. These experiments provide the first direct evidence that in addition to a mechanical role and relocalization of the DAPC, dystrophin and utrophin are able to alter both structural and biochemical properties of skeletal muscle. In addition, these mice provide unique insights into skeletal muscle fiber type composition.     

Redefinition of Dystrophin Isoform Distribution in Mouse Tissue by RT-PCR Implies Role in Nonmuscle Manifestations of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by a defect in a 427-kDa membrane-associated protein: dystrophin. The DMD gene also encodes several shorter isoforms which are believed to participate in nonmuscle manifestations of DMD, including abnormal retinal electrophysiology, dilated cardiomyopathy, mental retardation, and hearing defects. The purpose of this work was to determine the normal tissue expression of full-length dystrophin (Dp427) and the dystrophin isoforms Dp260, Dp140, Dp116, and Dp71, to aid in understanding what roles these isoforms might play in DMD nonmuscle manifestations. RT-PCR was performed on mRNA isolated from wild-type C57BL/6J mouse tissues, including brain, cardiac muscle, eye, intestine, kidney, liver, lung, skeletal muscle, spleen, stomach, testis, thymus, and uterus. RT-PCR amplification demonstrated that the isoforms were in a number of tissues which had not been revealed by previous Western and Northern blot analyses. Dp427 was expressed at equal levels in all tissues. Dp260 and Dp140 were present in all tissues tested, but the levels of expression varied. Dp116 was expressed in a subset of tissues and levels of expression varied. Dp71 was constitutively expressed in all tissues, suggesting that this isoform plays a basic role in normal tissue function. The expanded tissue distribution supports the hypothesis that dystrophin isoforms serve essential and unique functions, necessitating further investigation into their potential roles in DMD nonmuscle manifestations.     

Effects of dystrophin isoforms on signal transduction through neural retina: genotype-phenotype analysis of duchenne muscular dystrophy mouse mutants

Duchenne and Becker muscular dystrophy patients have mutations in the dystrophin gene. Most show reduced b-wave amplitudes in the dark-adapted electroretinogram (ERG). We studied normal C57BL/6J mice and five X-linked muscular dystrophy strains with different dystrophin mutations to determine whether the location of the mutation within the gene affects the mouse ERG and to correlate such effects with dystrophin isoform expression. Amplitudes and implicit times were measured for a-waves, b-waves, and digitally filtered oscillatory potentials. mdx and mdxCv5 mice, with mutations near the amino terminus and lacking expression of Dp427, had ERGs similar to those of C57BL/6J mice. mdxCv2 and mdxCv4 mice, with mutations in the center of dystrophin and who do not express isoforms Dp427, Dp260, or Dp140 (mdxCv4), had increased b-wave and oscillatory potential implicit times. mdxCv3 mice, with a mutation near the carboxy terminus resulting in deficiency of all dystrophin isoforms, had increased b-wave and oscillatory potential implicit times and reduced scotopic b-wave amplitudes. Fitting the a-wave data to a transduction activation phase mathematical model showed normal responses for all phenotypes, suggesting that the b-wave delays are due to defects beyond the rod outer segment, most likely at the rod to on-bipolar cell synapse. The variation in the ERG phenotype with the position of the dystrophin gene mutation suggests that there are different contributions by each isoform to retinal electrophysiology. Although Dp427 and Dp140 isoforms do not appear to be important contributors to the ERG, lack of Dp260 and possibly Dp71 isoforms is associated with an abnormal ERG.     

Dp260 disrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of beta-dystroglycan in the outer plexiform layer of the retina

Dp260 is a C-terminal isoform of dystrophin and is expressed specifically in the retina. Abnormal electroretinograms (ERG) in some Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients are likely linked to a disruption of Dp260. To clarify the importance of Dp260 in the retina, we examined dystrophin exon 52 knock- out mice, whose expression of Dp260 is impaired. We also confirmed the localization of Dp260 in the outer plexiform layer (OPL) of the retina. Disruption of Dp260 causes a change in the localization of beta- dystroglycan, which is normally found in the OPL of the retina. This suggests a requirement for Dp260 for normal formation of the dystrophin- dystroglycan complex in the retina. Dp71, also expressed in the retina, was, however, not detected in the OPL. The difference in localization of Dp260 and Dp71 implies that the two isoforms have different functions. The dystrophin exon 52 knock-out mice had a prolonged implicit time of the b-wave in ERG, although no significant change was observed in amplitude. These ERG findings differed from those of DMD and BMD patients, especially with regard to amplitude of the b-wave, but make it clear that Dp260 is required for normal electrophysiology.      

Reduced levels of dystrophin associated proteins in the brains of mice deficient for Dp71

Duchenne muscular dystrophy (DMD) is a progressive degenerative lethal muscle disease. A significant proportion of DMD affected children suffer also from mental retardation. The rod shaped protein, dystrophin, which is absent from or defective in the muscle of DMD patients, binds to a number of membrane associated proteins (known collectively as dystrophin associated proteins [DAPs]). The levels of DAPs is greatly reduced in the muscle of DMD patients and mdx mice, which lack dystrophin. In addition to dystrophin isoforms, the DMD gene codes also for several smaller proteins. One of the small proteins, Dp71, is expressed in most or all non-muscle tissues and is the major DMD gene product in the brain. The function of the small DMD gene products is unknown. Here we show that mutant mice which do not express the smaller non-muscle products of the DMD gene have a reduced level of DAPs in their brain. This suggests that Dp71 is important for the formation and/or stabilization of a DAPs complex in brain.      

Localization of dystrophin isoform Dp71 to the inner limiting membrane of the retina suggests a unique functional contribution of Dp71 in the retina

The electroretinograms (ERGs) of patients with Duchenne muscular dystrophy and an allelic variant of the mdx mouse (mdxCv3) have been shown to be abnormal. Analysis of five allelic variants of the mdx mouse with mutations in the dystrophin gene has shown that there is a correlation between the position of the mutation and the severity of the ERG abnormality. Three isoforms are expressed in the retina: Dp427, Dp260 and Dp71. Using indirect immunofluorescence and isoform-specific antibodies on retinal sections from three allelic mdx mouse strains, we have examined the localization of each of the isoforms. We show that Dp71 expression does not overlap with Dp427 and Dp260 expression at the outer plexiform layer (OPL). Instead, Dp71 is localized to the inner limiting membrane (ILM) and to retinal blood vessels. Moreover, we show that Dp260 and Dp71 differ structurally at their respective C-termini. In addition, we find that the proper localization of the beta- dystroglycan is dependent upon both Dp260 at the OPL and Dp71 expression at the ILM. Thus, Dp260 and Dp71 are non-redundant isoforms that are located at different sites within the retina yet have a common interaction with beta-dystroglycan. Our data suggest that both Dp71 and Dp260 contribute distinct but essential roles to retinal electrophysiology.      

A housekeeping type promoter, located in the 3' region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene

The 70.8 kDa protein product of the distal part of the giantDuchenne muscular dystrophy (DMD) gene, Dp71, is expressed inmany cell types and tissues. Anchored PCR, primer extensionand functional analysis of transfected constructs were usedto determine the 5' end of the mRNA and characterize the promoterof this major DMD gene product. The 5' untranslated region (5'UTR)of Dp71 is transcribed from a single exon; the promoter doesnot contain a TATA box, and has a very high GC content and severalpotential Sp1 binding sites. It is located more than 2000 kb3' to the muscle and brain type dystrophin promoters and only150 kb from the 3' end of the gene, suggesting that in mostDMD patients the expression of Dp71 Is unaffected.     

Expression of the dystrophin isoform Dp116 preserves functional muscle mass and extends lifespan without preventing dystrophy in severely dystrophic mice

Dp116 is a non-muscle isoform of dystrophin that assembles the dystrophin-glycoprotein complex (DGC), but lacks actin-binding domains. To examine the functional role of the DGC, we expressed the Dp116 transgene in mice lacking both dystrophin and utrophin (mdx:utrn(-/-)). Unexpectedly, expression of Dp116 prevented the most severe aspects of the mdx:utrn(-/-) phenotype. Dp116:mdx:utrn(-/-) transgenic mice had dramatic improvements in growth, mobility and lifespan compared with controls. This was associated with increased muscle mass and force generating capacity of limb muscles, although myofiber size and specific force were unchanged. Conversely, Dp116 had no effect on dystrophic injury as determined by muscle histopathology and serum creatine kinase levels. Dp116 also failed to restore normal fiber-type distribution or the post-synaptic architecture of the neuromuscular junction. These data demonstrate that the DGC is critical for growth and maintenance of muscle mass, a function that is independent of the ability to prevent dystrophic pathophysiology. Likewise, this is the first demonstration in skeletal muscle of a positive functional role for a dystrophin protein that lacks actin-binding domains. We conclude that both mechanical and non-mechanical functions of dystrophin are important for its role in skeletal muscle.