Steven J. Burden

The Receptor Tyrosine Kinase MuSK Is Required for Neuromuscular Junction Formation In Vivo

Formation of neuromuscular synapses requires a series of inductive interactions between growing motor axons and differentiating muscle cells, culminating in the precise juxtaposition of a highly specialized nerve terminal with a complex molecular structure on the postsynaptic muscle surface. The receptors and signaling pathways mediating these inductive interactions are not known. We have generated mice with a targeted disruption of the gene encoding MuSK, a receptor tyrosine kinase selectively localized to the postsynaptic muscle surface. Neuromuscular synapses do not form in these mice, suggesting a failure in the induction of synapse formation. Together with the results of an accompanying manuscript, our findings indicate that MuSK responds to a critical nerve-derived signal (agrin), and in turn activates signaling cascades responsible for all aspects of synapse formation, including organization of the postsynaptic membrane, synapse-specific transcription, and presynaptic differentiation.

Agrin Acts via a MuSK Receptor Complex

Formation of th neuromuscular junction depends upon reciprocal inductive interactions between the developing nerve and muscle, resulting in the precise juxtaposition of a differentiated nerve terminal with a highly specialized patch on the muscle membrane, termed the motor endplate. Agrin is a nerve-derived factor that can induced molecular reorganizations at the motor endplate, but the mechanism of action of agrin remains poorly understood. MuSK is a receptor tyrosine kinase localized to the motor endplate, seemingly well positioned to receive a key nerve-derived signal. Mice lacking either agrin or MuSK have recently been generated and exhibit similarly profound defects in their neuromuscular junctions. Here we demonstrate that agrin acts via a receptor complex that includes MuSK as well as a myotube-specific accessory component.

Lrp4 Is a Receptor for Agrin and Forms a Complex with MuSK

Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.

Patterning of Muscle Acetylcholine Receptor Gene Expression in the Absence of Motor Innervation

The patterning of skeletal muscle is thought to depend upon signals provided by motor neurons. We show that AChR gene expression and AChR clusters are concentrated in the central region of embryonic skeletal muscle in the absence of innervation. Neurally derived Agrin is dispensable for this early phase of AChR expression, but MuSK, a receptor tyrosine kinase activated by Agrin, is required to establish this AChR prepattern. The zone of AChR expression in muscle lacking motor axons is wider than normal, indicating that neural signals refine this muscle-autonomous prepattern. Neuronal Neuregulin-1, however, is not involved in this refinement process, nor indeed in synapse-specific AChR gene expression. Our results demonstrate that AChR expression is patterned in the absence of innervation, raising the possibility that similarly prepatterned muscle-derived cues restrict axon growth and initiate synapse formation.

Increased mitochondrial mass in mitochondrial myopathy mice

We have generated an animal model for mitochondrial myopathy by disrupting the gene for mitochondrial transcription factor A (Tfam) in skeletal muscle of the mouse. The knockout animals developed a myopathy with ragged-red muscle fibers, accumulation of abnormally appearing mitochondria, and progressively deteriorating respiratory chain function in skeletal muscle. Enzyme histochemistry, electron micrographs, and citrate synthase activity revealed a substantial increase in mitochondrial mass in skeletal muscle of the myopathy mice. Biochemical assays demonstrated that the increased mitochondrial mass partly compensated for the reduced function of the respiratory chain by maintaining overall ATP production in skeletal muscle. The increased mitochondrial mass thus was induced by the respiratory chain deficiency and may be beneficial by improving the energy homeostasis in the affected tissue. Surprisingly, in vitro experiments to assess muscle function demonstrated that fatigue development did not occur more rapidly in myopathy mice, suggesting that overall ATP production is sufficient. However, there were lower absolute muscle forces in the myopathy mice, especially at low stimulation frequencies. This reduction in muscle force is likely caused by deficient formation of force-generating actin–myosin cross bridges and/or disregulation of Ca2+ homeostasis. Thus, both biochemical measurements of ATP-production rate and in vitro physiological studies suggest that reduced mitochondrial ATP production might not be as critical for the pathophysiology of mitochondrial myopathy as thought previously.

A Dystroglycan Mutation Associated with Limb-Girdle Muscular Dystrophy

Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192→Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.

Neuregulins and their receptors.

The recent identification of an activator for the ErbB2/Neu receptor has uncovered a new family of polypeptide growth factors that undoubtedly play a major role in the regulation of neuronal growth and differentiation. These factors, called the neuregulins, are expressed in neural and mesenchymal tissues, and activate members of the epidermal growth factor family of receptor tyrosine kinases. The identification and characterization of the neuregulins and their receptors will facilitate the dissection of the biochemical pathways regulating nervous system development.

Crosslinking of proteins in acetylcholine receptor-rich membranes: Association between the β-subunit and the 43 kd subsynaptic protein

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four subunits (alpha, beta, gamma, delta) of the acetylcholine receptor (AChR) and for polypeptides at 43 kd and 270 kd. Reaction of these membranes with 3H-N-ethylmaleimide (3H-NEM) demonstrates that most of the available free sulfhydryls reside on the 43 kd protein. Cross-linking reagents that contain NEM as one reactive group, and N-hydroxysuccinimide as the other, were used to study the topography of the 43 kd protein in AChR-rich membranes. Proteins from cross-linked membranes were resolved by SDS-PAGE and the composition of crosslinked products was determined by Western blots and monoclonal antibodies. A crosslinked product at 110 kd was labeled by a monoclonal antibody to the beta-subunit and by a monoclonal antibody to the 43 kd protein, but not by monoclonal antibodies to the alpha, gamma, or delta subunits. The 110 kd crosslink was not produced in the presence of 10 mM lithium diiodosalicylate, which dissociates the 43 kd protein from the membrane. Thus the 43 kd protein is intimately associated with the AChR and in close proximity to the beta-subunit.

Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors.

Abstract The number, distribution, and stability of skeletal muscle acetylcholine receptors during development of the neuromuscular junction in the chick embryo were studied. The distribution and turnover of receptors labeled with 125 I-labeled α-bungarotoxin were determined by quantitative autoradiography on individual teased muscle fibers. Each posterior latissimus dorsi muscle fiber, which in the adult is singly innervated, has a high density of acetylcholine receptors at a single spot from embryonic Day 10 through hatching. The spots stain more intensely than elsewhere for acetylcholinesterase and are assumed to be end plates. The receptors at these spots are presumed to be junctional receptors. The junctional receptor density remains constant at 10 4 /μm 2 from embryonic Day 14 through adult life, although the area of the junction increases 40-fold. In contrast, the extrajunctional receptor density drops precipitously from 250/μm 2 on Day 14 to only 10/μm 2 on Day 19. This decrease in extrajunctional receptor density can be prevented by chronic paralysis with curare. The rate of autoradiographic grain loss from junctional and extrajunctional regions after a pulse injection of 125 I-labeled α-bungarotoxin indicates that both classes of embryonic receptors turn over at the same rate ( t 1 2 ⋍ 30 hr ).

Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses.

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse‐specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG‐stimulated signaling pathway is important for synapse‐specific gene expression.