Michael J. Ferns

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997)J. Biol. Chem. 272, 8117–8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24–48 h. Under these conditions, both muscle- and nerve-derived agrin increased the activity of β-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5′ from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic proteins. Together, these observations are therefore relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction and for the potential use of utrophin as a therapeutic strategy to counteract the effects of Duchenne muscular dystrophy.

Agrin regulates rapsyn interaction with surface acetylcholine receptors, and this underlies cytoskeletal anchoring and clustering

The acetylcholine receptor (AChR)-associated protein rapsyn is essential for neuromuscular synapse formation and clustering of AChRs, but its mode of action remains unclear. We have investigated whether agrin, a key nerve-derived synaptogenic factor, influences rapsyn-AChR interactions and how this affects clustering and cytoskeletal linkage of AChRs. By precipitating AChRs and probing for associated rapsyn, we found that in denervated diaphragm rapsyn associates with synaptic as well as with extrasynaptic AChRs showing that rapsyn interacts with unclustered AChRs in vivo. Interestingly, synaptic AChRs are associated with more rapsyn suggesting that clustering of AChRs may require increased interaction with rapsyn. In similar experiments in cultured myotubes, rapsyn interacted with intracellular AChRs and with unclustered AChRs at the cell surface, although surface interactions are much more prominent. Remarkably, agrin induces recruitment of additional rapsyn to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased rapsyn-AChR interaction and AChR clustering. Finally, time course experiments revealed that the agrin-induced increase occurs with AChRs that become cytoskeletally linked, and that this precedes receptor clustering. Thus, we propose that neural agrin controls postsynaptic aggregation of the AChR by enhancing rapsyn interaction with surface AChRs and inducing cytoskeletal anchoring and that this is an important precursor step for AChR clustering.

Agrin plays an organizing role in the formation of sympathetic synapses

Agrin is a nerve-derived factor that directs neuromuscular synapse formation, however its role in regulating interneuronal synaptogenesis is less clear. Here, we examine agrins role in synapse formation between cholinergic preganglionic axons and sympathetic neurons in the superior cervical ganglion (SCG) using agrin-deficient mice. In dissociated cultures of SCG neurons, we found a significant decrease in the number of synapses with aggregates of presynaptic synaptophysin and postsynaptic neuronal acetylcholine receptor among agrin-deficient neurons as compared to wild-type neurons. Moreover, the levels of pre- and postsynaptic markers at the residual synapses in agrin-deficient SCG cultures were also reduced, and these defects were rescued by adding recombinant neural agrin to the cultures. Similarly, we observed a decreased matching of pre- and postsynaptic markers in SCG of agrin-deficient embryos, reflecting a decrease in the number of differentiated synapses in vivo. Finally, in electrophysiological experiments, we found that paired-pulse depression was more pronounced and posttetanic potentiation was significantly greater in agrin-deficient ganglia, indicating that synaptic transmission is also defective. Together, these findings indicate that neural agrin plays an organizing role in the formation and/or differentiation of interneuronal, cholinergic synapses.

LG2 agrin mutation causing severe congenital myasthenic syndrome mimics functional characteristics of non-neural (z−) agrin

We describe a severe form of congenital myasthenic syndrome (CMS) caused by two heteroallelic mutations: a nonsense and a missense mutation in the gene encoding agrin (AGRN). The identified mutations, Q353X and V1727F, are located at the N-terminal and at the second laminin G-like (LG2) domain of agrin, respectively. A motor-point muscle biopsy demonstrated severe disruption of the architecture of the neuromuscular junction (NMJ), including: dispersion and fragmentation of endplate areas with normal expression of acetylcholinesterase; simplification of postsynaptic membranes; pronounced reduction of the axon terminal size; widening of the primary synaptic cleft; and, collection of membranous debris material in the primary synaptic cleft and in the subsynaptic cytoplasm. Expression studies in heterologous cells revealed that the Q353X mutation abolished expression of full-length agrin. Moreover, the V1727F mutation decreased agrin-induced clustering of the acetylcholine receptor (AChR) in cultured C2 muscle cells by >100-fold, and phosphorylation of the MuSK receptor and AChR beta subunit by ~tenfold. Surprisingly, the V1727F mutant also displayed increased binding to α-dystroglycan but decreased binding to a neural (z+) agrin-specific antibody. Our findings demonstrate that agrin mutations can associate with a severe form of CMS and cause profound distortion of the architecture and function of the NMJ. The impaired ability of V1727F agrin to activate MuSK and cluster AChRs, together with its increased affinity to α-dystroglycan, mimics non-neural (z−) agrin and are important determinants of the pathogenesis of the disease.

Identification of a motif in the acetylcholine receptor β subunit whose phosphorylation regulates rapsyn association and postsynaptic receptor localization

At the neuromuscular junction, the acetylcholine receptor (AChR) is specifically clustered in the postsynaptic membrane via interactions with rapsyn and other scaffolding proteins. However, it remains unclear where these proteins bind on the AChR and how the interactions are regulated. Here, we define a phosphorylation-dependent binding site on the receptor that mediates agrin-induced clustering. Using chimeric proteins in which CD4 is fused to the large intracellular loop of each of the AChR subunits we found that agrin induced clustering of only chimeras containing the β subunit loop. By making deletions in the β loop we defined a 20 amino-acid sequence that is sufficient for clustering. The sequence contains a conserved tyrosine (Y390) whose phosphorylation is induced by agrin and whose mutation abolished clustering of β loop chimeras and their ability to inhibit agrin-induced clustering of the endogenous AChR. Phosphorylation of the AChR β subunit is correlated with increased rapsyn/AChR binding, suggesting that the effect of βY390 phosphorylation on clustering is mediated by rapsyn. Indeed, we found that rapsyn associated with CD4-β loop chimeras in a phosphorylation-dependent manner, and that agrin increased the ratio of rapsyn binding to wild type AChR but not to AChR-β3F/3F, which lacks β loop tyrosine phosphorylation sites. Together, these findings suggest that agrin-induced phosphorylation of the β subunit motif increases the stoichiometry of rapsyn binding to the AChR, thereby helping to stably cluster the receptor and anchor it at high density in the postsynaptic membrane.

Agrin-independent activation of the agrin signal transduction pathway.

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle-specific kinase (MuSK) and the AChR-beta subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition.

Acute Severe Animal Model of Anti–Muscle-Specific Kinase Myasthenia: Combined Postsynaptic and Presynaptic Changes

OBJECTIVES To determine the pathogenesis of anti-muscle-specific kinase (MuSK) myasthenia, a newly described severe form of myasthenia gravis associated with MuSK antibodies characterized by focal muscle weakness and wasting and absence of acetylcholine receptor antibodies, and to determine whether antibodies to MuSK, a crucial protein in the formation of the neuromuscular junction (NMJ) during development, can induce disease in the mature NMJ. Design, Setting, and PARTICIPANTS Lewis rats were immunized with a single injection of a newly discovered splicing variant of MuSK, MuSK 60, which has been demonstrated to be expressed primarily in the mature NMJ. Animals were assessed clinically, serologically, and by repetitive stimulation of the median nerve. Muscle tissue was examined immunohistochemically and by electron microscopy. RESULTS Animals immunized with 100 μg of MuSK 60 developed severe progressive weakness starting at day 16, with 100% mortality by day 27. The weakness was associated with high MuSK antibody titers, weight loss, axial muscle wasting, and decrementing compound muscle action potentials. Light and electron microscopy demonstrated fragmented NMJs with varying degrees of postsynaptic muscle end plate destruction along with abnormal nerve terminals, lack of registration between end plates and nerve terminals, local axon sprouting, and extrajunctional dispersion of cholinesterase activity. CONCLUSIONS These findings support the role of MuSK antibodies in the human disease, demonstrate the role of MuSK not only in the development of the NMJ but also in the maintenance of the mature synapse, and demonstrate involvement of this disease in both presynaptic and postsynaptic components of the NMJ.

Challenging the neurocentric view of neuromuscular synapse formation.

heterologous cells appears to be able to mobilize surface Precision of synaptic connectivity is essential for proper AChRs into small clusters. Subsequently, these small clusfunction of the nervous system. This is achieved by the ters of AChRs coalesce into synaptic densities by anchorprojection of axons to correct targets within a region of ing to a transmembrane complex of dystrophin-associated the nervous system as well as by synapse formation on proteins assembling in the postsynaptic membrane and discrete domains of individual cells. This precision is extracellular matrix to help anchor AChE. In addition to this widely thought to involve molecules that guide axons sequence of postynaptic events, contact of motoneuron to their targets, followed by a period of experiential fine growth cones with their targets also stops axonal growth tuning of synaptic connectivity. The recent past has and induces differentiation of nerve terminals. Interestbeen rife with new information about the molecular maingly, mice null for agrin and especially for MuSK have chinery of synapses, though work on the neuromuscular exuberant axonal growth as well as reduced nerve terminal junction (NMJ) has provided the conceptual framework differentiation. Thus, this ligand-receptor pair is necessary for study of the more complex synapses in the brain. to induce a retrograde signal from muscle that regulates In particular, these studies have provided a detailed axon growth and presynaptic differentiation. understanding of the assembly of the postsynaptic apA later phase of synaptogenesis involves specific reguparatus of NMJs. lation of gene expression in the synaptic region to consoliEven a casual observer would notice that NMJs are date the earlier steps in synapse formation. For example, commonly found within a narrow band in the midline of ongoing release of acetylcholine from differentiating nerve skeletal muscles. More detailed investigation reveals terminals activates AChRs in the muscle and triggers electhat these synapses are exquisitely organized with posttrical activity. Propagation of this activity can downregusynaptic membrane folds that are aligned with vesicle late the expression of AChR genes within all nuclei of the release sites in the nerve terminal. Acetylcholine recepmyofiber (Burden, 1998; Sanes and Lichtman, 1999). In tors (AChRs) are found at the tops of the folds and, the face of this, neuregulin/glial growth factor from the together with acetylcholinesterase (AChE) in the extranerve and muscle may bind to and activate the erB-2/4 cellular matrix, are tightly restricted to the 0.1% of the family of receptor tyrosine kinases, which in turn would muscle cell surface occupied by the synapse. Indeed, increase transcription of AChR genes. These more conthe NMJ constitutes a structure, on an otherwise uniform ventional receptor tyrosine kinases increase the transcripmyotube, that extends from the nerve terminal, through tion of AChR genes. Activation could also contribute to the synaptic extracellular matrix, to postsynaptic muscle spatial restriction of AChR transcription since neuregulin membrane, muscle cytoskeleton, and even the subsywould become concentrated within synaptic basal lamina naptic nuclei that specifically express mRNAs for variety and erb receptors are localized in subsynaptic plasma of synapse-specific genes. membrane. A host of studies in culture and in vivo support neural It is fair to say that this neurocentric model of synapse control of neuromuscular synapse formation (Burden, formation does not take fully into account studies of Harris 1998; Sanes and Lichtman, 1999). One particularly vivid and coworkers (Braithwaite and Harris, 1979; Harris, 1981) example comes from studies of nerve muscle coculwho had shown that developing muscle rendered aneural tures. Aneural muscle cells develop clusters of AChRs, with neurotoxins still expressed AChRs in a localized rebut when neurons are added to muscle cultures, growth gion of the muscle. In a recent paper, Burden and coworkcones typically ignore these clusters to innervate other ers (Yang et al., 2000) confirmed and extended these data regions of the cell (Anderson and Cohen, 1977; Frank in a series of insightful studies of mice null for the topoand Fischbach, 1979). The noninnervated clusters of isomerase IIb gene. In these mice, motor nerves extended AChRs disperse during this process. Subsequent work to the vicinity of developing skeletal muscles but failed to has identified a number of molecules key to the asseminvade the diaphragm and innervate the myofibers. These bly and stabilization of the postsynaptic specialization. workers found, however, that AChRs were found in clusA widely accepted scenario (Figure 1A), supported by ters localized to the midline of the muscle, suggesting both in vivo and in vitro data, would have the motoneuthat muscle is patterned in the absence of innervation.

Rapsyn interacts with the muscle acetylcholine receptor via α-helical domains in the α, β, and ε subunit intracellular loops

At the developing vertebrate neuromuscular junction, the acetylcholine receptor becomes aggregated at high density in the postsynaptic muscle membrane. Receptor localization is regulated by the motoneuron-derived factor, agrin, and requires an intracellular, scaffolding protein called rapsyn. However, it remains unclear where rapsyn binds on the acetylcholine receptor and how their interaction is regulated. In this study, we identified rapsyns binding site on the acetylcholine receptor using chimeric constructs where the intracellular domain of CD4 was substituted for the major intracellular loop of each mouse acetylcholine receptor subunit. When expressed in heterologous cells, we found that rapsyn clustered and cytoskeletally anchored CD4-alpha, beta and epsilon subunit loops but not CD4-delta loop. Rapsyn-mediated clustering and anchoring was highest for beta loop, followed by epsilon and alpha, suggesting that rapsyn interacts with the loops with different affinities. Moreover, by making deletions within the beta subunit intracellular loop, we show that rapsyn interacts with the alpha-helical region, a secondary structural motif present in the carboxyl terminal portion of the subunit loops. When expressed in muscle cells, rapsyn co-immunoprecipitated together with a CD4-alpha helical region chimera, independent of agrin signaling. Together, these findings demonstrate that rapsyn interacts with the acetylcholine receptor via an alpha-helical structural motif conserved between the alpha, beta and epsilon subunits. Binding at this site likely mediates the critical rapsyn interaction involved in localizing the acetylcholine receptor at the neuromuscular junction.

Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

At the developing vertebrate neuromuscular junction, postsynaptic localization of the acetylcholine receptor (AChR) is regulated by agrin signaling via the muscle specific kinase (MuSK) and requires an intracellular scaffolding protein called rapsyn. In addition to its structural role, rapsyn is also necessary for agrin-induced tyrosine phosphorylation of the AChR, which regulates some aspects of receptor localization. Here, we have investigated the molecular mechanism by which rapsyn mediates AChR phosphorylation at the rodent neuromuscular junction. In a heterologous COS cell system, we show that MuSK and rapsyn induced phosphorylation of beta subunit tyrosine 390 (Y390) and delta subunit Y393, as in muscle cells. Mutation of beta Y390 or delta Y393 did not inhibit MuSK/rapsyn-induced phosphorylation of the other subunit in COS cells, and mutation of beta Y390 did not inhibit agrin-induced phosphorylation of the delta subunit in Sol8 muscle cells; thus, their phosphorylation occurs independently, downstream of MuSK activation. In COS cells, we further show that MuSK-induced phosphorylation of the beta subunit was mediated by rapsyn, as MuSK plus rapsyn increased beta Y390 phosphorylation more than rapsyn alone and MuSK alone had no effect. Intriguingly, MuSK also induced tyrosine phosphorylation of rapsyn itself. We then used deletion mutants to map the rapsyn domains responsible for activation of cytoplasmic tyrosine kinases that phosphorylate the AChR subunits. We found that rapsyn C-terminal domains (amino acids 212-412) are both necessary and sufficient for activation of tyrosine kinases and induction of cellular tyrosine phosphorylation. Moreover, deletion of the rapsyn RING domain (365-412) abolished MuSK-induced tyrosine phosphorylation of the AChR beta subunit. Together, these findings suggest that rapsyn facilitates AChR phosphorylation by activating or localizing tyrosine kinases via its C-terminal domains.