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Markus A Ruegg

Publications and source records attributed to Markus A Ruegg.

11 recordsLinked to original sources
PubMed2005

Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering.

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.

Agrin↗
PubMed2005

Tyrosine phosphatase regulation of MuSK-dependent acetylcholine receptor clustering.

During vertebrate neuromuscular junction (NMJ) development, nerve-secreted agrin induces acetylcholine receptor (AChR) clustering in muscle by activating the muscle-specific tyrosine kinase MuSK. Recently, it has been recognized that MuSK activation-dependent AChR clustering occurs in embryonic muscle even in the absence of agrin, but how this process is regulated is poorly understood. We report that inhibition of tyrosine phosphatases in cultured C2 mouse myotubes using pervanadate enhanced MuSK auto-activation and agrin-independent AChR clustering. Moreover, phosphatase inhibition also enlarged the AChR clusters induced by agrin in these cells. Conversely, in situ activation of MuSK in cultured Xenopus embryonic muscle cells, either focally by anti-MuSK antibody-coated beads or globally by agrin, stimulated downstream tyrosine phosphatases, which could be blocked by pervanadate treatment. Immunoscreening identified Shp2 as a major tyrosine phosphatase in C2 myotubes and down-regulation of its expression by RNA interference alleviated tyrosine phosphatase suppression of MuSK activation. Significantly, depletion of Shp2 increased both agrin-independent and agrin-dependent AChR clustering in myotubes. Our results suggest that muscle tyrosine phosphatases tightly regulate MuSK activation and signaling and support a novel role of Shp2 in MuSK-dependent AChR clustering.

Agrin↗
PubMed2005

Structural and functional diversity generated by alternative mRNA splicing.

Alternative mRNA splicing is becoming increasingly recognized as an important mechanism for the generation of structural and functional diversity in proteins. Recent estimations predict that approximately 50% of all eukaryotic proteins can be alternatively spliced. Several lines of evidence suggest that alternative mRNA splicing results in small changes in protein structure and is likely to fine-tune the function and specificity of the affected protein. However, knowledge of how alternative splicing regulates cellular processes on the molecular level is still limited. It is only recently that structures of alternatively spliced proteins have been solved. These studies have shown that alternative splicing affects the structure not only in the vicinity of the splice site but also at long distance.

Alternative Splicing↗
PubMed2005

Overexpression of mini-agrin in skeletal muscle increases muscle integrity and regenerative capacity in laminin-alpha2-deficient mice.

Mutations in the gene encoding the alpha2 subunit of laminins cause the severe "merosin-deficient congenital muscular dystrophy" (MDC1A). We have recently shown that overexpression of a miniaturized form of the molecule agrin (mini-agrin) counteracts the disease in dy(W)/dy(W) mice, a model for MDC1A. However, these mice express some residual truncated laminin-alpha2, suggesting that the observed amelioration might be due to mini-agrin's presenting the residual laminin-alpha2 to its receptors. Here we show that the mini-agrin counteracts the disease in dy(3K)/dy(3K) mice, which are null for laminin-alpha2. As in dy(W)/dy(W) mice, mini-agrin improves both the function and structure of muscle. We show that muscle regeneration after injury is severely impaired in dy(3K)/dy(3K) mice but is restored in the mini-agrin-expressing littermates. In summary, our results 1) show that the direct linkage of muscle basal lamina with the sarcolemma is the basis of mini-agrin-mediated amelioration and 2) provide unprecedented evidence that this linkage is important for proper regeneration of muscle fibers after injury. Our findings thus suggest that treatment with mini-agrin might be beneficial over the entire spectrum of the MDC1A disease, whose severity inversely correlates with expression levels and the size of the truncation in laminin-alpha2.

Agrin↗
PubMed2004

Inhibition of synapse assembly in mammalian muscle in vivo by RNA interference.

The formation of the vertebrate neuromuscular junction (NMJ) requires the receptor tyrosine kinase MuSK and the adaptor molecule rapsyn. Here, we report that the phenotypes of mice deficient in these two molecules can be reproduced by RNA interference (RNAi) in rat muscle in vivo. Specifically, double-stranded RNA (dsRNA) targeting MuSK and rapsyn inhibited the formation of the NMJ in rat muscle fibres in vivo, while dsRNA targeting nonessential proteins did not have any effect. Moreover, plasmids that trigger RNAi to MuSK induced the disassembly of existing NMJs. These results thus demonstrate for the first time the functionality of dsRNA in silencing endogenous genes in adult mammalian muscle in vivo. Moreover, they show that MuSK is also required for the maintenance of the NMJ, offering a mechanistic explanation for the myasthenia gravis caused by auto-antibodies to MuSK.

Animals↗
PubMed2004

Modulation of agrin function by alternative splicing and Ca2+ binding.

The aggregation of acetylcholine receptors on postsynaptic membranes is a key step in neuromuscular junction development. This process depends on alternatively spliced forms of the proteoglycan agrin with "B-inserts" of 8, 11, or 19 residues in the protein's globular C-terminal domain, G3. Structures of the neural B8 and B11 forms of agrin-G3 were determined by X-ray crystallography. The structure of G3-B0, which lacks inserts, was determined by NMR. The agrin-G3 domain adopts a beta jellyroll fold. The B insert site is flanked by four loops on one edge of the beta sandwich. The loops form a surface that corresponds to a versatile interaction interface in the family of structurally related LNS proteins. NMR and X-ray data indicate that this interaction interface is flexible in agrin-G3 and that flexibility is reduced by Ca(2+) binding. The plasticity of the interaction interface could enable different splice forms of agrin to select between multiple binding partners.

Agrin↗
PubMed2003

Expression of mouse agrin in normal, denervated and dystrophic muscle.

Agrin is a heparan sulfate proteoglycan that is required for the development of postsynaptic specializations at the neuromuscular junction. An alternatively spliced isoform of agrin that lacks this activity is found in basement membranes of several tissues including embryonic muscle. Overexpression of a miniaturized form of this agrin isoform ameliorates the severe muscle dystrophy of laminin alpha2-deficient mice, a mouse model for merosin-deficient congenital muscle dystrophy. Several lines of evidence indicate that this amelioration is based on the high-affinity binding of the mini-agrin to the laminins and to alpha-dystroglycan. Here, we used antibodies raised against mouse agrin to evaluate protein expression in adult muscle of normal and dystrophic mice. We find that expression of agrin in non-synaptic region varies greatly between different muscles in wild-type mice and that its levels are altered in dystrophic muscle.

Agrin↗
PubMed2003

New insights into the roles of agrin.

The heparan sulphate proteoglycan agrin is expressed as several isoforms in various tissues. Agrin is best known as a crucial organizer of postsynaptic differentiation at the neuromuscular junction, but it has recently also been implicated in the formation of the immunological synapse, the organization of the cytoskeleton and the amelioration of function in diseased muscle. So the activities of agrin might be of broader significance than previously anticipated.

Agrin↗
PubMed2002

An intrinsic distinction in neuromuscular junction assembly and maintenance in different skeletal muscles.

We analyzed the formation of neuromuscular junctions (NMJs) in individual muscles of the mouse embryo. Skeletal muscles can be assigned to one of two distinct classes of muscles, termed "Fast Synapsing" (FaSyn) and "Delayed Synapsing" (DeSyn) muscles, which differ significantly with respect to the initial focal clustering of postsynaptic AChRs, the timing of presynaptic maturation, and the maintenance of NMJs in young adult mice. Differences between classes were intrinsic to the muscles and manifested in the absence of innervation or agrin. Paralysis or denervation of young adult muscles resulted in disassembly of AChR clusters on DeSyn muscles, whereas those on FaSyn muscles were preserved. Our results show that postsynaptic differentiation processes intrinsic to FaSyn and DeSyn muscles influence the formation of NMJs during development and their maintenance in the adult.

Agrin↗
PubMed2002

A newly identified chromosomal microdeletion and an N-box mutation of the AChR epsilon gene cause a congenital myasthenic syndrome.

Congenital myasthenic syndromes (CMSs) are frequently caused by mutations of the coding region of the acetylcholine receptor epsilon subunit (AChRepsilon) gene leading to a reduced expression of the acetylcholine receptor (AChR) at the postsynaptic membrane. Two recent observations have linked two different N-box mutations of the human AChRepsilon promoter to a clinical CMS phenotype. N-boxes are regulatory sequence elements of mammalian promoters that confer synapse-specific expression of several genes, including the AChR subunit genes. Here, we report on a novel point mutation (epsilon-154G-->A) in the N-box of the AChRepsilon promoter in a German CMS pedigree. Semiquantitative analysis of AChRepsilon mRNA levels in the patient's muscle indicated significantly impaired AChRepsilon expression. We provide additional evidence of a pathogenic role for this mutation using the mutated promoter (epsilon-154G-->A) driving a heterologous gene (luciferase) in rat skeletal muscle. We show that agrin-induced gene expression is significantly reduced by the N-box mutant (mt) compared with the wild-type (wt) promoter. Refined haplotype analysis and direct sequencing revealed maternal inheritance of the mutant AChRepsilon promoter (epsilon-154G-->A) together with paternal inheritance of a chromosomal microdeletion (Delta1290 bp) encompassing the promoter and the first two exons of the AChRepsilon gene in the index patient. In conclusion, we provide genetic and functional evidence that a mutation of the AChRepsilon subunit promoter (epsilon-154G-->A) causes CMS due to the reduction of gene expression in skeletal muscle. Moreover, this is the first report of a chromosomal microdeletion affecting an AChR gene. This type of mutation may be missed in standard screening techniques of CMS patients.

Adult↗