Faculty Bibliography

Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2+/- mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation.

Skeletal myogenesis is regulated by signal transduction, but the factors and mechanisms involved are not well understood. The group I Paks Pak1 and Pak2 are related protein kinases and direct effectors of Cdc42 and Rac1. Group I Paks are ubiquitously expressed and specifically required for myoblast fusion in Drosophila We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice has no overt effect on skeletal muscle development or regeneration. However, combined muscle-specific deletion of Pak1 and Pak2 results in reduced muscle mass and a higher proportion of myofibers with a smaller cross-sectional area. This phenotype is exacerbated after repair to acute injury. Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling pathway.

Motor axons approach muscles that are prepatterned in the prospective synaptic region. In mice, prepatterning of acetylcholine receptors requires Lrp4, a LDLR family member, and MuSK, a receptor tyrosine kinase. Lrp4 can bind and stimulate MuSK, strongly suggesting that association between Lrp4 and MuSK, independent of additional ligands, initiates prepatterning in mice. In zebrafish, Wnts, which bind the Frizzled (Fz)-like domain in MuSK, are required for prepatterning, suggesting that Wnts may contribute to prepatterning and neuromuscular development in mammals. We show that prepatterning in mice requires Lrp4 but not the MuSK Fz-like domain. In contrast, prepatterning in zebrafish requires the MuSK Fz-like domain but not Lrp4. Despite these differences, neuromuscular synapse formation in zebrafish and mice share similar mechanisms, requiring Lrp4, MuSK, and neuronal Agrin but not the MuSK Fz-like domain or Wnt production from muscle. Our findings demonstrate that evolutionary divergent mechanisms establish muscle prepatterning in zebrafish and mice.

ALS is a devastating disease, progressing from detachment of motor nerve terminals to paralytic, lethal respiratory failure within several years of diagnosis. The mechanisms responsible for axon withdrawal are poorly understood, but the loss of neuromuscular synapses is sufficient to cause muscle paralysis and therefore central to the disease. Although the subsequent loss of motor neurons has received more attention, preventing or delaying motor neuron cell death without preserving neuromuscular synapses cannot stop disease progression. Skeletal muscles provide retrograde signals that promote the differentiation and stabilization of motor nerve terminals (1, 2). The production of retrograde signals depends upon a synaptic receptor tyrosine kinase, termed MuSK, and Lrp4, a receptor for Agrin that forms a complex with MuSK (3). Because a failure to maintain neuromuscular synapses is central to all forms of ALS, we tested whether increasing retrograde signalling in SOD1^G93A transgenic mice would stabilize neuromuscular synapses, delay axon withdrawal and ameliorate disease symptoms (4). We found that a modest increase in MuSK expression is sufficient to maintain neuromuscular synapses in SOD1^G93A mice, delaying muscle denervation and improving muscle function for over one month (4). Thus, the loss of motor nerve terminals can be delayed by co-opting a retrograde signalling pathway that normally functions to stimulate the differentiation of these terminals (4). These findings suggest a novel therapeutic approach to slow the steady decline in motor function in ALS. Moreover, because motor axon withdrawal is an early, characteristic and critical feature of disease in all forms of ALS, increasing MuSK activity might provide benefit in both familial and sporadic forms of ALS. We sought a more practical therapeutic approach to activate MuSK in vivo. A previous study reported that two human single chain variable region antibodies (ScFv) to MuSK, as well as IgG molecules reconstituted from these ScFv antibodies, stimulate MuSK in cultured myotubes (5). Thus, these antibodies provide an attractive means to activate MuSK in vivo. We found that a single injection of a humanized agonist antibody to MuSK substantially reduced denervation and increased innervation for one month. Thus, increasing MuSK activity, after denervation and disease symptoms were evident, slows synaptic loss. We are currently studying whether the agonist antibody improves motor function and whether chronic dosing with a murinized agonist antibody preserves synapses, improves motor performance, reduces motor neuron cell death and prolongs longevity of SOD1^G93A mice

Lrp4, the muscle receptor for neuronal Agrin, is expressed in the hippocampus and areas involved in cognition. The function of Lrp4 in the brain, however, is unknown, as Lrp4-/- mice fail to form neuromuscular synapses and die at birth. Lrp4-/- mice, rescued for Lrp4 expression selectively in muscle, survive into adulthood and showed profound deficits in cognitive tasks that assess learning and memory. To learn whether synapses form and function aberrantly, we used electrophysiological and anatomical methods to study hippocampal CA3-CA1 synapses. In the absence of Lrp4, the organization of the hippocampus appeared normal, but the frequency of spontaneous release events and spine density on primary apical dendrites were reduced. CA3 input was unable to adequately depolarize CA1 neurons to induce long-term potentiation. Our studies demonstrate a role for Lrp4 in hippocampal function and suggest that patients with mutations in Lrp4 or auto-antibodies to Lrp4 should be evaluated for neurological deficits.

The formation and maintenance of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. As a consequence, acetylcholine receptors (AChRs) become highly concentrated in the postsynaptic membrane and arranged in perfect register with active zones in the presynaptic nerve terminal, insuring for rapid, robust and reliable synaptic transmission. During development, motor axons approach and recognize muscle that is primed, or prepatterned in the prospective synaptic region. Muscle prepatterning is established by MuSK, a receptor tyrosine kinase, and Lrp4, a member of the LDLR family. Lrp4 associates with MuSK and stimulates MuSK kinase activity, increasing Lrp4 and MuSK expression and causing the clustering of Lrp4 and MuSK. Once clustered, Lrp4 functions as a direct retrograde signal for presynaptic differentiation, causing motor axons to stop growing and develop specializations required for neurotransmitter release. Nascent synapses are stabilized by neuronal Agrin, which is released by motor nerve terminals and binds to Lrp4, stimulating further association between Lrp4 and MuSK and increasing MuSK kinase activity. Lrp4 thus has a central role in coordinating synaptic differentiation, as Lrp4 not only binds Agrin and stimulates postsynaptic differentiation but also acts in turn as a direct retrograde signal for presynaptic differentiation. Mutations in Agrin, Lrp4 and MuSK, as well as additional genes that function in this signaling pathway, cause congenital myasthenia, and auto-antibodies to Lrp4, MuSK, or AChRs are responsible for myasthenia gravis. I will summarize experiments that have contributed to this model of neuromuscular synapse formation, indicate how this knowledge has provided insight into causes for neuromuscular disease, and describe a therapeutic approach for preserving synapses and treating neuromuscular diseases

Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options.

Muscle-specific kinase (MuSK) is essential for each step in neuromuscular synapse formation. Before innervation, MuSK initiates postsynaptic differentiation, priming the muscle for synapse formation. Approaching motor axons recognize the primed, or prepatterned, region of muscle, causing motor axons to stop growing and differentiate into specialized nerve terminals. MuSK controls presynaptic differentiation by causing the clustering of Lrp4, which functions as a direct retrograde signal for presynaptic differentiation. Developing synapses are stabilized by neuronal Agrin, which is released by motor nerve terminals and binds to Lrp4, a member of the low-density lipoprotein receptor family, stimulating further association between Lrp4 and MuSK and increasing MuSK kinase activity. In addition, MuSK phosphorylation is stimulated by an inside-out ligand, docking protein-7 (Dok-7), which is recruited to tyrosine-phosphorylated MuSK and increases MuSK kinase activity. Mutations in MuSK and in genes that function in the MuSK signaling pathway, including Dok-7, cause congenital myasthenia, and autoantibodies to MuSK, Lrp4, and acetylcholine receptors are responsible for myasthenia gravis.

Amyotrophic lateral sclerosis (ALS) is a devastating disease that progresses from detachment of motor nerve terminals to complete muscle paralysis and lethal respiratory failure within 5 years of diagnosis. Genetic studies have linked mutations in several genes to ALS, and mice bearing mutations in SOD1 recapitulate hallmark features of the disease. We investigated whether disease symptoms can be ameliorated by co-opting the retrograde signaling pathway that promotes attachment of nerve terminals to muscle. We crossed SOD1G93A mice with transgenic mice that express MuSK, a receptor tyrosine kinase that is required for retrograde signaling, and we used histological and behavioral assays to assess motor innervation and behavior. A 3-fold increase in MuSK expression delayed the onset and reduced the extent of muscle denervation, improving motor function for more than a month without altering survival. These findings suggest that increasing MuSK activity by pharmacological means has the potential to improve motor function in ALS.