Primary adhalinopathy: a common cause of autosomal recessive muscular dystrophy of variable severity.

Marked deficiency of muscle adhalin, a 50 kDa sarcolemmal dystrophin-associated glycoprotein, has been reported in severe childhood autosomal recessive muscular dystrophy (SCARMD). This is a Duchenne-like disease affecting both males and females first described in Tunisian families. Adhalin deficiency has been found in SCARMD patients from North Africa Europe, Brazil, Japan and North America (SLR & KPC, unpublished data). The disease was initially linked to an unidentified gene on chromosome 13 in families from North Africa, and to the adhalin gene itself on chromosome 17q in one French family in which missense mutations were identified. Thus there are two kinds of myopathies with adhalin deficiency: one with a primary defect of adhalin (primary adhalinopathies), and one in which absence of adhalin is secondary to a separate gene defect on chromosome 13. We have examined the importance of primary adhalinopathies among myopathies with adhalin deficiency, and describe several additional mutations (null and missense) in the adhalin gene in 10 new families from Europe and North Africa. Disease severity varies in age of onset and rate of progression, and patients with null mutations are the most severely affected.

Clustering and immobilization of acetylcholine receptors by the 43-kD protein: a possible role for dystrophin-related protein.

Recombinant acetylcholine receptors (AChRs) expressed on the surface of cultured fibroblasts become organized into discrete membrane domains when the 43-kD postsynaptic protein (43k) is co-expressed in the same cells (Froehner, S.C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410; Phillips, W. D., M. C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we show that AChRs present on the fibroblast cell surface prior to transfection of 43k are recruited into 43k-rich membrane domains. Aggregated AChRs show increased resistance to extraction with Triton X-100, suggesting a 43k-dependent linkage to the cytoskeleton. Myotubes of the mouse cell line C2 spontaneously display occasional AChR/43k-rich membrane domains that ranged in diameter up to 15 microns, but expressed many more when 43k was overexpressed following transfection of 43k cDNA. However, the membrane domains induced by recombinant 43k were predominantly small (< or = 2 microns). We were then interested in whether the cytoskeletal component, dystrophin related protein (DRP; Tinsley, J. M., D. J. Blake, A. Roche, U. Fairbrother, J. Riss, B. C. Byth, A. E. Knight, J. Kendrick-Jones, G. K. Suthers, D. R. Love, Y. H. Edwards, and K. E. Davis, 1992. Nature (Lond.). 360:591-593) contributed to the development of AChR clusters. Immunofluorescent anti-DRP staining was present at the earliest stages of AChR clustering at the neuromuscular synapse in mouse embryos and was also concentrated at the large AChR-rich domains on nontransfected C2 myotubes. Surprisingly, anti-DRP staining was concentrated mainly at the large, but not the small AChR clusters on C2 myotubes suggesting that DRP may be principally involved in permitting the growth of AChR clusters.

Non-muscle alpha-dystroglycan is involved in epithelial development.

The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. Here we show that dystroglycan during mouse development in nonmuscle tissues is expressed in epithelium. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two was previously shown for laminin alpha 1 chain. Immunofluorescence showed that dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturbed development of epithelium in kidney organ culture, whereas control antibodies did not do so. We suggest that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha-dystroglycan to E3 fragment of laminin-1.

Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex.

The 43 kDa AChR-associated protein rapsyn is required for the clustering of nicotinic acetylcholine receptors (AChRs) at the developing neuromuscular junction, but the functions of other postsynaptic proteins colocalized with the AChR are less clear. Here we use a fibroblast expression system to investigate the role of the dystrophin-glycoprotein complex (DGC) in AChR clustering. The agrin-binding component of the DGC, dystroglycan, is found evenly distributed across the cell surface when expressed in fibroblasts. However, dystroglycan colocalizes with AChR-rapsyn clusters when these proteins are coexpressed. Furthermore, dystroglycan colocalizes with rapsyn clusters even in the absence of AChR, indicating that rapsyn can cluster dystroglycan and AChR independently. Immunofluorescence staining using a polyclonal antibody to utrophin reveals a lack of staining of clusters, suggesting that the immunoreactive species, like the AChR, does not mediate the observed rapsyndystroglycan interaction. Rapsyn may therefore be a molecular link connecting the AChR to the DGC. At the neuromuscular synapse, rapsyn-mediated linkage of the AChR to the cytoskeleton-anchored DGC may underlie AChR cluster stabilization.

Abnormal expression of laminin suggests disturbance of sarcolemma-extracellular matrix interaction in Japanese patients with autosomal recessive muscular dystrophy deficient in adhalin.

Dystrophin is associated with several novel sarcolemmal proteins, including a laminin-binding extracellular glycoprotein of 156 kD (alpha-dystroglycan) and a transmembrane glycoprotein of 50 kD (adhalin). Deficiency of adhalin characterizes a severe autosomal recessive muscular dystrophy prevalent in Arabs. Here we report for the first time two mongoloid (Japanese) patients with autosomal recessive muscular dystrophy deficient in adhalin. Interestingly, adhalin was not completely absent and was faintly detectable in a patchy distribution along the sarcolemma in our patients. Although the M and B2 subunits of laminin were preserved, the B1 subunit was greatly reduced in the basal lamina surrounding muscle fibers. Our results raise a possibility that the deficiency of adhalin may be associated with the disturbance of sarcolemma-extracellular matrix interaction leading to sarcolemmal instability.

The expression of dystrophin-associated glycoproteins during skeletal muscle degeneration and regeneration. An immunofluorescence study.

The distribution and expression of dystrophin and three of the dystrophin-associated glycoproteins (DAG), alpha-dystroglycan (156 kDa DAG), beta-dystroglycan (43 kDa DAG) and adhalin (50 kDa DAG) in rat skeletal muscle were studied during a controlled cycle of degeneration and regeneration induced by the injection of a snake venom. Cryosections of muscle at various stages of degeneration and regeneration were labeled using monoclonal antibodies to the three glycoproteins and examined at fixed time points after venom injection. Adhalin and alpha-dystroglycan remained present at the sarcolemma throughout the entire cycle of degeneration and regeneration. beta-Dystroglycan, on the other hand, was lost from the sarcolemma by 12 hours and reappeared 2 days after venom injection when new muscle fibers were being formed. Dystrophin was not lost from the sarcolemma until 24 hours after venom injection and did not reappear at the membrane until 4 days. It is suggested that dystrophin and the glycoprotein complex are synthesized separately, both temporally and spatially, and only become associated at the plasma membrane during the later stages of regeneration. The expression of beta-dystroglycan in the regenerating muscle fibers was first seen at sites of newly forming plasma membrane that were closely associated with the old basal lamina tube. The basal lamina may therefore have a regulatory or modulatory role in the expression of the DAG.

Adhalin mRNA and cDNA sequence are normal in the cardiomyopathic hamster.

Adhalin is deficient in two forms of human muscular dystrophy, one due to mutations in the adhalin gene and one linked to an unidentified gene on chromosome 13. Because adhalin is deficient in skeletal and cardiac muscles of BIO 14.6 hamsters, which experience both myopathy and cardiomyopathy, cDNA encoding adhalin from BIO 14.6 hamster skeletal muscle was cloned and sequenced. Adhalin mRNA was expressed at normal levels in BIO 14.6 hamster cardiac muscle, and no mutation in adhalin coding sequence was found, indicating that the inherited myopathy and cardiomyopathy of the BIO 14.6 hamster are most likely not due to mutations in the adhalin gene.

Developmental expression of cloned cardiac potassium channels.

Cardiac K+ channels are responsible for repolarization of the action potential and are the targets of several antiarrhythmic drugs. This study examines the differential expression of six K+ channel mRNAs during rat heart development. RK1 and RK2 K+ channel transcripts were undetectable prior to 10 days after birth. In contrast, RK4 mRNA was present at equivalent levels from day 14 in utero to 20 days after birth. RK3 and RK5 were detected as early as 14 days in utero. These data indicate that K+ channel expression in the heart is closely regulated and further argue for physiologically distinct roles for K+ channel isoforms.

Functional characterization of RK5, a voltage-gated K+ channel cloned from the rat cardiovascular system.

A voltage-sensitive K+ channel previously cloned from rat heart designated RK5 (rat Kv4.2) (Roberds and Tamkun, 1991, Proc. Natl. Acad. Sci. USA 88, 1798-1802) was functionally characterized in the Xenopus oocyte expression system. RK5 is a homolog of the Drosophila Shal K+ channel, activates with a rise time of 2.8 ms, has a midpoint for activation of -1 mV and rapidly inactivates with time constants of 15 and 60 ms. RK5 is sensitive to 4-AP, IC50 = 5 mM, and is insensitive to TEA and dendrotoxins. The voltage dependence and kinetics of the RK5 induced currents suggest this channel contributes to the Ito current in heart.

Alpha-dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy.

The dystrophin-glycoprotein complex was examined in dystrophin-deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin-associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of alpha-dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.

Applying genomics tools to identify therapeutic targets for asthma.

Asthma is a chronic inflammatory disease of the airways, the susceptibility to which is strongly influenced by genetics. Genomics, the study of the human genome, is redefining the process for rapidly identifying novel therapeutic targets for asthma and other diseases. One approach is to search for polymorphisms in genes that increase susceptibility to the disease in order to identify genes and cellular pathways relevant to the disease process. In asthma, for example, regardless of the genetic factors that contribute to susceptibility, good drug targets could be found that affect epithelial integrity, allergic response, and the recruitment or activity of inflammatory cells. Such targets may consist of proteins that are specifically expressed in certain cell types, proteins whose expression is regulated during the disease process or proteins involved in the destructive process. This review discusses some of the genomics tools that can be used to identify new molecular targets, which in turn are useful in screening for novel compounds likely to affect diseases such as asthma.

Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart.

Five distinct K+ channel cDNA molecules (RK1 to RK5) were cloned from either rat heart or rat aorta cDNA libraries. Four of the channels, RK1 to RK4, are similar or identical to Shaker-like K+ channels previously identified in rat brain cDNA libraries. Major differences among RK1 to RK4 exist in the amino- and carboxyl-terminal regions and in amino acids representing potential extracellular sequence between the S1 and S2 hydrophobic domains. RK5 encodes a unique channel of 490 amino acids having six hydrophobic domains but only five basic residues in the putative voltage-sensing domain. Unlike RK1 to RK4, RK5 is a rat homologue of the Drosophila Shal family of K+ channels, which have not been previously described in mammals. Although RK5 mRNA is present in cardiac atrium and ventricle, it is most abundant in brain. RK1, RK2, and RK3 transcripts are predominantly found in brain but are present also at lower levels in other tissues, such as heart and aorta. RK2 is absent from skeletal muscle whereas RK1 and RK3 are present in this tissue. RK4 mRNA is ubiquitous in electrically excitable tissue, being present at comparable levels in atrium, ventricle, aorta, brain, and skeletal muscle. The cloning of RK5 confirms the presence in mammals of all four Drosophila K+ channel families: Shaker, Shab, Shaw, and Shal.

Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel.

The interaction of quinidine with a cloned human cardiac potassium channel (HK2) expressed in a stable mouse L cell line was studied using the whole-cell tight-seal voltage-clamp technique. Quinidine (20 microM) did not affect the initial sigmoidal activation time course of the current. However, it reduced the peak current and induced a subsequent decline, with a time constant of 8.2 +/- 0.8 msec, to 28 +/- 6% of control (at +60 mV). The concentration dependence of HK2 block at +60 mV yielded an apparent KD of 6 microM and a Hill coefficient of 0.9. The degree of block was voltage dependent. Block increased from 0.60 +/- 0.09 at 0 mV to 0.72 +/- 0.06 at +60 mV with 20 microM quinidine and from 0.39 +/- 0.20 to 0.48 +/- 0.16 with 6 microM. Paired analysis in seven experiments with 20 microM quinidine indicated that the voltage-dependent increase in block was significant (difference, 12 +/- 4%; p less than 0.001). This voltage dependence was described by an equivalent electrical distance delta of 0.19 +/- 0.02, which suggested that at the binding site quinidine experienced 19% of the applied transmembrane electrical field, referenced to the inner surface. Quinidine reduced the tail current amplitude and slowed the time course relative to control, resulting in a "crossover" phenomenon. These data indicate that 1) the charged form of quinidine blocks the HK2 channel after it opens, 2) binding occurs within the transmembrane electrical field (probably in or near the ion permeation pathway), and 3) unbinding is required before the channel can close.

Primary structure and muscle-specific expression of the 50-kDa dystrophin-associated glycoprotein (adhalin).

The 50-kDa dystrophin-associated glycoprotein (50-DAG) is a component of the dystrophin-glycoprotein complex, which links the muscle cytoskeleton to the extracellular matrix. 50-DAG is specifically deficient in skeletal muscle of patients with severe childhood autosomal recessive muscular dystrophy and in skeletal and cardiac muscles of BIO 14.6 cardiomyopathic hamsters. The lack of 50-DAG leads to a disruption and dysfunction of the dystrophin-glycoprotein complex in these diseases. The cDNA encoding 50-DAG has now been cloned from rabbit skeletal muscle. The 50-DAG deduced amino acid sequence predicts a novel protein having 387 amino acids, a 17-amino acid signal sequence, one transmembrane domain, and two potential sites of N-linked glycosylation. Affinity-purified antibodies against rabbit 50-DAG fusion proteins or synthetic peptides specifically recognized a 50-kDa protein in skeletal muscle sarcolemma and the 50-kDa component of the dystrophin-glycoprotein complex. In contrast to dystroglycan, which is expressed in a wide variety of muscle and non-muscle tissues, 50-DAG is expressed only in skeletal and cardiac muscles and in selected smooth muscles. Finally, 50-DAG mRNA is present in mdx and Duchenne muscular dystrophy (DMD) muscle, indicating that the down-regulation of this protein in DMD and the mdx mouse is likely a post-translational event.

Ultrastructural localization of adhalin, alpha-dystroglycan and merosin in normal and dystrophic muscle.

Adhalin and alpha-dystroglycan are two components of a complex of proteins that, in conjunction with dystrophin, provide a link between the subsarcolemmal cytoskeleton and the basal lamina of the extracellular matrix of skeletal muscle. In the absence of dystrophin, in Duchenne muscular dystrophy (DMD) and the mdx mouse, levels of adhalin, alpha-dystroglycan and other components of the complex, are severely reduced, and it has been speculated that this might be an important factor in precipitating myofibre necrosis. However, there is, as yet, little information on how these proteins interact structurally or functionally. From biochemical data it might be predicted that adhalin and alpha-dystroglycan are positioned more peripherally in the muscle cell than dystrophin and more proximal than merosin. Using single and double immunogold labelling we here show that adhalin is localized to the plasma membrane with the majority of the gold probe particles situated on the membrane's outer face, while alpha-dystroglycan labelling is seen on material which projects from the outer face and which, in places, forms strands that stretch to the basal lamina. When double labelling of laminin and alpha-dystroglycan is carried out, laminin is localized to the proximal face of the basal lamina, facing the alpha-dystroglycan. In DMD the labelling of adhalin and alpha-dystroglycan is severely reduced quantitatively (although the vestige that remains is positioned normally) but merosin is expressed normally, showing that its incorporation is independent of that of dystrophin and its associated proteins.

A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering.

Synapse formation is characterized by the accumulation of molecules at the site of contact between pre- and postsynaptic cells. Agrin, a protein implicated in the regulation of this process, causes the clustering of acetylcholine receptors (AChRs). Here we characterize an agrin-binding site on the surface of muscle cells, show that this site corresponds to alpha-dystroglycan, and present evidence that alpha-dystroglycan is functionally related to agrin activity. Furthermore, we demonstrate that alpha-dystroglycan and adhalin, components of the dystrophin-associated glycoprotein complex, as well as utrophin, colocalize with agrin-induced AChR clusters. Thus, agrin may function by initiating or stabilizing a synapse-specific membrane cytoskeleton that in turn serves as a scaffold upon which synaptic molecules are concentrated.

Effect of the antiviral compound MDL 20,610 on some aspects of murine immune function.

At physiologically relevant concentrations an antiviral compound should not perturb the host's ability to mount an immune response against the infecting virus or some other opportunistic pathogen. The purpose of this study was to evaluate the immunomodulatory activity of the antiviral compound MDL 20,610 using murine models. When tested in vitro at the limit of aqueous solubility (6 microM), MDL 20,610 has no significant effect on neutrophil function as assessed by cell migration against FMLP and LTB4 gradients, myeloperoxidase secretion or 0.-2 production. In addition, 6 microM MDL 20,610 has no significant effect on macrophage function as determined by 0.-2 production, Ia and Mac-1 antigen expression and expression of Fc gamma receptors. Finally, MDL 20,610 does not significantly affect in vivo (1-100 mg/kg/day) NK cell activity or DTH to oxazolone; but treatment of mice with 50 or 100 mg MDL 20,610/kg/day significantly (P less than 0.01) enhances SRBC IgM antibody synthesis. These data indicate that MDL 20,610 is relatively devoid of immunomodulatory activity.

A 5' dystrophin duplication mutation causes membrane deficiency of alpha-dystroglycan in a family with X-linked cardiomyopathy.

5'-mutations in the dystrophin gene can result in cardiomyopathy without clinically-apparent skeletal myopathy. The effect of dystrophin mutations on the assembly and stability of the dystrophin associated protein (DAP) complex in human heart are not fully understood. The molecular defect in the dystrophin complex was explored in a family with an X-linked pedigree and severe dilated cardiomyopathy. Dystrophin gene analysis demonstrated a 5' duplication involving exons 2-7, which encodes the N-terminal actin binding domain of dystrophin. Ribonuclease protection and PCR assays demonstrated a reduction in muscle promoter transcribed dystrophin mRNA in the heart compared to skeletal muscle. A deficiency of cardiac dystrophin protein was observed by Western blot and lack of membrane localization by immunocytochemistry. The cardiac expression of the dystrophin related protein utrophin was increased, and the 43 kDa (beta-dystroglycan), 50 kDa (alpha-sarcoglycan) and 59 kDa (syntrophin) dystrophin associated proteins (DAPs) were co-isolated and present in nearly normal amounts in the membrane. However, cardiac dystrophin deficiency and increased utrophin expression were associated with loss of extracellular 156 kDa dystrophin associated glycoprotein (alpha-dystroglycan) binding to the cardiomyocyte membrane. alpha-Dystroglycan is responsible for linkage of the dystrophin complex to the extracellular matrix protein laminin. Therefore, 5' dystrophin mutations can reduce cardiac dystrophin mRNA, protein expression, and dystrophin function in X-linked cardiomyopathy (XLCM). The presence of membrane-associated beta-dystroglycan, alpha-sarcoglycan, syntrophin, and utrophin are insufficient to maintain cardiac function. This XLCM family has a 5' dystrophin gene mutation resulting in cardiac dystrophin deficiency and a loss of alpha-dystroglycan membrane binding.

Chromosomal mapping in the mouse of eight K(+)-channel genes representing the four Shaker-like subfamilies Shaker, Shab, Shaw, and Shal.

The four Shaker-like subfamilies of Shaker-, Shab-, Shaw-, and Shal-related K+ channels in mammals have been defined on the basis of their sequence homologies to the corresponding Drosophila genes. Using interspecific backcrosses between Mus musculus and Mus spretus, we have chromosomally mapped in the mouse the Shaker-related K(+)-channel genes Kcna1, Kcna2, Kcna4, Kcna5, and Kcna6; the Shab-related gene Kcnb1; the Shaw-related gene Kcnc4; and the Shal-related gene Kcnd2. The following localizations were determined: Chr 2, cen-Acra-Kcna4-Pax-6-a-Pck-1-Kras-3-Kcn b1 (corresponding human Chrs 11p and 20q, respectively); Chr 3, cen-Hao-2-(Kcna2, Kcnc4)-Amy-1 (human Chr 1); and Chr 6, cen-Cola-2-Met-Kcnd2-Cpa-Tcrb-adr/Clc-1-Hox-1.1-Myk - 103-Raf-1-(Tpi-1, Kcna1, Kcna5, Kcna6) (human Chrs 7q and 12p, respectively). Thus, there is a cluster of at least three Shaker-related K(+)-channel genes on distal mouse Chr 6 and a cluster on Chr 2 that at least consists of one Shaker-related and one Shaw-related gene. The three other K(+)-channel genes are not linked to each other. The map positions of the different types of K(+)-channel genes in the mouse are discussed in relation to those of their homologs in man and to hereditary diseases of mouse and man that might involve K+ channels.

Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord.

Mutations of copper,zinc-superoxide dismutase (cu,zn SOD) are found in patients with a familial form of amyotrophic lateral sclerosis. When expressed in transgenic mice, mutant human cu,zn SOD causes progressive loss of motor neurons with consequent paralysis and death. Expression profiling of gene expression in SOD1-G93A transgenic mouse spinal cords indicates extensive glial activation coincident with the onset of paralysis at 3 months of age. This is followed by activation of genes involved in metal ion regulation (metallothionein-I, metallothionein-III, ferritin-H, and ferritin-L) at 4 months of age just prior to end-stage disease, perhaps as an adaptive response to the mitochondrial destruction caused by the mutant protein. Induction of ferritin-H and -L gene expression may also limit iron catalyzed hydroxyl radical formation and consequent oxidative damage to lipids, proteins, and nucleic acids. Thus, glial activation and adaptive responses to metal ion dysregulation are features of disease in this transgenic model of familial amyotrophic lateral sclerosis.