Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro

We have studied presynaptic and postsynaptic differentiation at neuromuscular junctions in vitro by examining the localization of synapse-specific proteins. In nerve–muscle co-cultures, the synaptic vesicle protein synaptotagmin (p65) accumulated in the nerve terminal overlying myotubes in association with postsynaptic cluster of acetylcholine receptors (AChRs), heparan sulfate proteoglycan (HSPG), laminin, and agrin. Inhibition of collagen synthesis with cis-hydroxyproline decreased the nerve-induced clustering of AChRs in muscle cells as well as that caused by exogenous agrin in muscle-only cultures. Moreover, accumulation of HSPG at contacts was also inhibited in cis-hydroxyproline–treated cultures. However, accumulation of p65 in nerve fibers at sites of muscle contact, a sign of presynaptic differentiation, was unaffected by cis-hydroxyproline treatment. In addition, even in cis-hydroxyproline–inhibited cultures, agrin was evident at more than 90% of contacts showing accumulation of p65 in the nerve terminal. Therefore, a mechanism exists to maintain agrin concentrations at nerve–muscle contacts, even when at least some extracellular matrix (ECM) proteins are disrupted. Our results suggest that HSPG is not required for the induction of nerve terminal differentiation but are consistent with the idea that HSPG or other ECM proteins are important in both nerve-and agrin-induced AChR clustering. In particular, agrin accumulation at sites of nerve–muscle contact is not sufficient to induce AChR clusters when the ECM at these contacts is disrupted. © 1995 John Wiley & Sons, Inc.     

Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions

alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.     

Acetylcholine receptor clustering associates with proteoglycan biosynthesis in C2 variant and heterkaryon muscle cells

Several lines of evidence have suggested roles for proteoglycans (PGs) in acetylcholine receptor (AChR) clustering on muscle cells. One line of evidence comes from the correlation between a defect in the biosynthesis of glycosaminoglycans (GAGs), the defining carbohydrates of PGs, and the failure of spontaneous AChR clustering in the S27 cell line, a genetic variant of the C2 muscle cell line. Two approaches were used in the present study to investigate whether GAG and AChR clustering defects are causally linked. First, the formation of AChR clusters was examined in two more variant lines, S11 and S26, also isolated from the C2 muscle cell line on the basis of deficiencies in GAG biosynthesis. S11 and S26, like S27, are also defective in AChR clustering. Ion exchange analysis of the GAGs made by the S11, S26, and S27 lines revealed that the defects in GAG biosynthesis differ between the three lines. Second, heterokaryon myotubes formed between pairs of the GAG defective variants were tested for complementation in both AChR clustering and GAG biosynthesis. AChR clusters were conspicuous on individual heterokaryon myotubes, and GAG biosynthesis was restored to near wild type levels in the heterokaryon cultures. Complementation in GAG biosynthesis corroborates the biochemical data that the relevant mutations in the genetic variants are in different genes and establishes that the defects are not dominant. The consistent correlation between GAG defects and the failure of AChR clustering across three independent genetic variants and the complementary association of GAG biosynthesis with AChR clustering in heterokaryon myotubes argues against a chance association of the two phenotypes and for a causal relationship between PGs and AChR clustering. A prominent chondroitin sulfate peak correlated with AChR clustering in the heterokaryon cultures. This is consistent with earlier results suggesting that chondroitin sulfate in general is required for the spontaneous clustering of AChRs in C2 cultures and further suggests that a particular chondroitin sulfate proteoglycan may be essential for the clustering process. © 1996 John Wiley & Sons, Inc.     

Protein 4.1R expression in normal and dystrophic skeletal muscle

4.1R pre-mRNA alternative splicing results in multiple mRNA and protein isoforms that are expressed in virtually all tissues. More specifically, isoforms containing the alternative exon 17a, are exclusively expressed in muscle tissues. In this report, we show that these isoforms are preferentially present in the myoplasm of fast myofibres. 4.1R epitopes are also found at the sarcolemma of both slow and fast myofibres in normal muscle. Interestingly, they are absent from dystrophin-deficient sarcolemma of DMD muscle, and colocalize with partially expressed dystrophin in BMD muscle. We also show that alternative splicing of exons 16 and 17a is regulated during muscle differentiation in an asynchronous fashion, with an early inclusion of exon 16 in forming myotubes, and a late inclusion of exon 17a. Consistently, Western blot analysis led to characterize mainly an approximately 96/98-kDa doublet bearing exons 16-17a-encoding peptide, exclusively occurring in the differentiated muscle.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.     

-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 -dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.     

α-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 α-dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.     

The apparent absence of lamin B1 and emerin in many tissue nuclei is due to epitope masking

Summary Immunolocalization studies have concluded that the nuclear membrane protein, emerin, is absent from many cell types and that lamin B1 is absent from adult heart and skeletal muscle. We now show that epitope masking in the nucleus is often responsible for failure to detect emerin and lamins in human, rat and pig tissues. Human heart cardiomyocyte nuclei were negative for lamin B1 using a commercial mAb, but were positive using two other lamin B1 antibodies, mAb8D1 and pAbB1-cbs. Rat hippocampal neuronal nuclei were immunostained by mAb8D1, but not pAbB1-cbs, while the commercial antibody stained only a subset. These data suggest that different regions of the lamin B1 molecule are masked in different tissues. Similarly, pig spleen had fewer emerin-positive nuclei than lung (5% vs. 32%), although their emerin content was similar by Western blotting. As mAbs against six epitopes gave the same result, the whole emerin molecule is either masked or redistributed in a subset of cells. Our findings argue that immunostaining evidence can be misleading for expression of nuclear envelope proteins. Problems with lamin B1 immunostaining can be avoided by using mAb8D1, but use of antibodies recognizing different epitopes may reveal cell-specific protein interactions in the nucleus.     

Key roles for the small leucine-rich proteoglycans in renal and pulmonary pathophysiology

Background

Small leucine-rich proteoglycans (SLRPs) are molecules that have signaling roles in a multitude of biological processes. In this respect, SLRPs play key roles in the evolution of a variety of diseases throughout the human body.

Scope of Review

We will critically review current developments in the roles of SLRPs in several types of disease of the kidney and lungs. Particular emphasis will be given to the roles of decorin and biglycan, the best characterized members of the SLRP gene family.

Major Conclusions

In both renal and pulmonary disorders, SLRPs are essential elements that regulate several pathophysiological processes including fibrosis, inflammation and tumor progression. Decorin has remarkable antifibrotic and antitumorigenic properties and is considered a valuable potential treatment of these diseases. Biglycan can modulate inflammatory processes in lung and renal inflammation and is a potential target in the treatment of inflammatory conditions.

General Significance

SLRPs can serve as either treatment targets or as potential treatment in renal or lung disease. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

The tomato gene Sw5 is a member of the coiled coil,nucleotide binding,leucine-rich repeat class of plant resistance genes and confers resistance to TSWV in tobacco

Tomato spotted wilt virus is an important threat to tomato production worldwide. A single dominant resistance gene locus, Sw5, originating from Lycopersicon peruvianum, has been identified and introgressed in cultivated tomato plants. Here we present the genomic organization of a 35 250 bp fragment of a BAC clone overlapping the Sw5 locus. Two highly homologous (95%) resistance gene candidates were identified within 40 kb of the CT220 marker. The genes, tentatively named Sw5-a and Sw5-b, encode proteins of 1245 and 1246 amino acids, respectively, and are members of the coiled-coil, nucleotide-binding-ARC, leucine-rich repeat group of resistance gene candidates. Promoter and terminator regions of the genes are also highly homologous. Both genes significantly resemble the tomato nematode and aphid resistance gene Mi and, to a lesser extent, Pseudomonas syringae resistance gene Prf. Transformation of Nicotiana tabacum cv. SR1 plants revealed that the Sw5-b gene, but not the Sw5-a gene, is necessary and sufficient for conferring resistance against tomato spotted wilt virus.     

The role of agrin in synaptic development,plasticity and signaling in the central nervous system

Development of the neuromuscular junction (NMJ) requires secretion of specific isoforms of the proteoglycan agrin by motor neurons. Secreted agrin is widely expressed in the basal lamina of various tissues, whereas a transmembrane form is highly expressed in the brain. Expression in the brain is greatest during the period of synaptogenesis, but remains high in regions of the adult brain that show extensive synaptic plasticity. The well-established role of agrin in NMJ development and its presence in the brain elicited investigations of its possible role in synaptogenesis in the brain. Initial studies on the embryonic brain and neuronal cultures of agrin-null mice did not reveal any defects in synaptogenesis. However, subsequent studies in culture demonstrated inhibition of synaptogenesis by agrin antisense oligonucleotides or agrin siRNA. More recently, a substantial loss of excitatory synapses was found in the brains of transgenic adult mice that lacked agrin expression everywhere but in motor neurons. The mechanisms by which agrin influences synapse formation, maintenance and plasticity may include enhancement of excitatory synaptic signaling, activation of the “muscle-specific” receptor tyrosine kinase (MuSK) and positive regulation of dendritic filopodia. In this article I will review the evidence that agrin regulates synapse development, plasticity and signaling in the brain and discuss the evidence for the proposed mechanisms.     

The expanding roles of the Sd/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2

Background

The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sd^a antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups.

Scope of review

We describe the multiple and unsuspected aspects of the biology of the Sd^a antigen and its biosynthetic enzyme β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings.

Major conclusions

The immunodominant sugar of the Sd^a antigen is a β1,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sd^a antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model.

General significance

The expression of the Sd^a antigen has a wide impact on the physiology and the pathology of different biological systems.