Expert Opinion on Therapeutic Targets

ISSN: 1472-8222 (Print) 1744-7631 (Online) Journal homepage: http://www.tandfonline.com/loi/iett20

Agrin-LRP4-MuSK signaling as a therapeutic target for myasthenia gravis and other neuromuscular disorders Kinji Ohno, Bisei Ohkawara & Mikako Ito To cite this article: Kinji Ohno, Bisei Ohkawara & Mikako Ito (2017): Agrin-LRP4-MuSK signaling as a therapeutic target for myasthenia gravis and other neuromuscular disorders, Expert Opinion on Therapeutic Targets, DOI: 10.1080/14728222.2017.1369960 To link to this article: http://dx.doi.org/10.1080/14728222.2017.1369960

Accepted author version posted online: 21 Aug 2017.

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Date: 22 August 2017, At: 04:53

Agrin-LRP4-MuSK signaling as a therapeutic target for myasthenia gravis and other neuromuscular disorders

Kinji Ohno1, Bisei Ohkawara1, Mikako Ito1

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Division of Neurogenetics, Nagoya University Graduate School of Medicine

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Correspondence to:

Kinji Ohno, MD, PhD, Division of Neurogenetics, Center for Neurological Diseases

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and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, ShowaTel: +81-52-744-2447 Fax: +81-52-744-2449

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Email: [email protected]

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ku, Nagoya 466-8550, Japan.

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Keywords

acetylcholine receptor, agrin, LRP4, MuSK, congenital myasthenic syndromes, myasthenia gravis

Abstract

Introduction: Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including myasthenia gravis, LambertEaton myasthenic syndrome, Isaacs’ syndrome, congenital myasthenic syndromes, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. Except for sarcopenia, all are orphan diseases. In addition, the NMJ signal transduction is impaired by tetanus, botulinum, curare, α-bungarotoxin, conotoxins,

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organophosphate, sarin, VX, and soman to name a few. Areas covered: This review covers the agrin-LRP4-MuSK signaling pathway, which

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transduction at the NMJ. We also address diseases caused by autoantibodies against

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the NMJ molecules and by germline mutations in genes encoding the NMJ molecules.

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Expert opinion: Representative small compounds to treat the defective NMJ signal transduction are cholinesterase inhibitors, which exert their effects by increasing the amount of acetylcholine at the synaptic space. Another possible therapeutic strategy

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to enhance the NMJ signal transduction to increase the number of AChRs, but no

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currently available drug has this functionality.

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drives clustering of acetylcholine receptors (AChRs) and ensures efficient signal

1. Introduction Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases (Fig. 1). Autoimmune NMJ diseases include myasthenia gravis (MG)1, Lambert-Eaton myasthenic syndrome2, and acquired neuromyotonia (Isaacs’ syndrome)3. In MG, autoantibodies are detected against acetylcholine receptor (AChR), muscle-specific receptor tyrosine kinase (MuSK)4-6, low-density lipoprotein (LDL) receptor-related protein 4 (LRP4)7-9, and agrin10, 11. Some MG

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patients may have autoantibodies only against clustered AChR, and not a single

AChR12. Among these autoantibodies, pathogenicity of autoantibodies against LRP4

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autoantibodies are the reduced number of AChRs at the motor endplate. In Lambert-

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Eaton myasthenic syndrome, autoantibodies are raised against the presynaptic P/Qtype voltage-gated calcium channel (VGCC, P/Q-type Ca2+ channel). In Isaacs’

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syndrome, autoantibody is raised against the presynaptic voltage-gated potassium channel (VGKC). In contrast to MG, autoantibodies to VGCC and VGKC do not cause AChR deficiency at the motor endplate. Inherited NMJ diseases are collectively

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referred to as congenital myasthenic syndromes (CMS), which include endplate acetylcholine receptor (AChR) deficiency, slow channel syndrome, fast channel

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syndrome, CMS with episodic apnea, endplate acetylcholinesterase (AChE) deficiency, and congenital Lambert-Eaton myasthenic syndrome13, 14. CMS caused by germline mutations in genes expressed at the motor endplate, as well as in genes

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encoding glycosylation enzymes, demonstrate AChR deficiency, whereas the other CMS subtypes show no AChR deficiency. Furthermore, the NMJ signal transduction is compromised in Fukuyama-type congenital muscular dystrophy15, amyotrophic

lateral sclerosis (ALS)16, and sarcopenia17-19, all of which are likely to be caused by AChR deficiency. Signal transduction at the NMJ is also affected by bacterial toxins

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and agrin remain to be validated. Shared features of MG caused by these

(tetanus and botulinum), alkaloid toxin (curare), snake toxin (α-bungarotoxin), snail toxins (conotoxins), and chemical toxins (organophosphates, sarin, VX, and soman et al.). Among these toxins, curare and α-bungarotoxin block ion channel opening of AChR. Representative small compounds to treat defective NMJ signal transduction due to AChR deficiency are cholinesterase inhibitors (physostigmine and pyridostigmine et al.), which exert their effects by increasing the number of acetylcholine (ACh) molecules at the synaptic space. Another possible therapeutic

strategy to enhance the NMJ signal transduction is to facilitate clustering of AChRs, but no currently available drug has this functionality.

2. Agrin-LRP4-MuSK signaling pathway to induce AChR clustering

2.1. Clustering of AChRs at the NMJ Clustering of AChRs at the NMJ is mediated by the agrin-LRP4-MuSK

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signaling (Figs. 1 and 2)20, 21. A membrane-spanning protein, LRP4, forms a dimer.

Another membrane-spanning protein, MuSK, also forms a dimer. Thus, a tetrameric

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endplate22. Neural agrin released from the nerve terminal of the spinal motor neurons

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(SMNs) binds to LRP4 and activates the kinase domain of MuSK to self-

phosphorylate MuSK. A number of intracellular signaling molecules have been

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individually analyzed, but the whole landscape of the intracellular signaling mechanisms remains to be elucidated (Fig. 2). Casein kinase 2 (CK2) also phosphorylates MuSK, and knockout of CK2 in mouse compromises AChR

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clustering23. Activated MuSK is internalized by endocytosis to exert its AChR clustering effect24. Activated MuSK recruits a non-catalytic adaptor protein Dok-725.

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The recruited Dok-7 further facilitates phosphorylation of MuSK. Activated MuSK phosphorylates two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins of Crk and Crk-L26, as well as their

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downstream molecules, Sorbs1 and Sorbs227. Activated MuSK also interacts with

Tid1 encoded by DNAJA3, which is required for Dok-7-induced AChR clustering, but is not required for MuSK phosphorylation28. Activated MuSK interacts with and activates the tyrosine kinase Abl29 and the geranylgeranyl transferase I (GGT)30, which leads to activation of Rho GTPases31, 32. Activated Rho GTPases then recruits

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LRP4-MuSK complex is formed as a heteromeric receptor for agrin at the motor

Pak1, which is a serine/threonine kinase bound to MuSK via Dvl33. The Rho GTPases also recruits WASP family proteins to activate the actin-related protein 2 and 3 (Arp2/3) complex, which nucleates new actin filaments34. Activated MuSK triggers association of AChR β subunit with the adenomatous polyposis coli protein, APC35. The C-terminal third of APC bundles actin filaments, and crosslinks actin filaments and microtubules, which is inhibited the microtubule plus-end tracking protein, EB136. Activated MuSK phosphorylates the SHC transforming protein, Shc4, which then tyrosine-phosphorylates AChR subunits37. MuSK-mediated activation of Src

family kinases also phosphorylates AChR subunits38, 39. Indeed, knockout of Src family kinases destabilizes AChR clusters at the NMJ40. Tyrosine-phosphorylation of the AChR β subunit markedly enhances cell surface expression of AChR41. MuSK phosphorylation and the subsequent activation of its downstream molecules stimulate a subsynaptic structural protein, rapsyn, to self-aggregate and induce anchoring of AChRs on the rapsyn scaffold42. Transcription of RAPSN encoding rapsyn is promoted by the nuclear factor kappaB (NF-kappaB)43. Rapsyn binds to the α, β, and

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ε subunits with the highest affinity for the β subunit44. Rapsyn also binds to β-

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Cortical actin cytoskeleton also serves as a scaffold for AChR49. Clustering of AChR

at the NMJ is mediated by the capture of microtubules through phosphorylation of the

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microtubule plus end-tracking protein, CLASP2, by the glycogen synthase kinase 3β, GSK3β50, 51. The phosphatidylinositol-3,4,5-triphosphate (PIP3)-binding protein,

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LL5β, binds to CLASP2 to induce AChR clustering52. Caveolin-3 associates with MuSK and AChR, and induces AChR clustering53. CHRNA1 mRNA encoding the

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AChR α subunit and the RNA-binding protein, StauI, bind to AChR to induce AChR clustering54. Interestingly, lack of the intermediate filament protein, nestin55, as well as lack of the serine-threonine kinase, Cdk556, increase AChR clusters. Calpain

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similarly induces dispersion of AChR clusters, and agrin enhances association of calpain and rapsyn to inhibit the AChR-dispersing activity of calpain57. Another

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inhibitor of AChR clustering is sodium nitrate in diet or environment58. Wnt ligands also bind to the Frizzled-like cysteine-rich domain (Fz-CRD) of

LRP4 and induce AChR clustering (Fig. 1)59. Binding of Wnt ligands to LRP4 may60

or may not61 be required for prepatterning of AChRs at the center of muscle fibers in development. Interestingly, one of the Wnt receptors, Frizzled-9, induces cytosolic

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dystroglycan and links the rapsyn scaffold to the subsynaptic cytoskeleton45-48.

accumulation of β-catenin, but impairs AChR clustering in skeletal muscle cells62 We recently reported that another SMN-derived molecule, Rspo2, binds to

Lgr5 at the motor endplate and enhances MuSK phosphorylation in an agrinindependent manner and to the level of ~80% of agrin-mediated MuSK phosphorylation (Fig. 1)63. Enhancement of the agrin-LRP4-MuSK signaling pathway

by small compounds or biological products is a promising therapeutic option for defective NMJ signal transduction.

2.2. Muscle nicotinic AChR Nicotinic AChRs are pentameric ligand-gated ion channels, which include four macromolecules: cationic AChRs, cationic serotonergic receptors (5HT3), anionic glycine receptors, and anionic GABAA and GABAC receptors64. Heteromeric neuronal nicotinic AChRs are made of combinations of α (α2-α10) and β subunits (β2-β4) with a stoichiometry of α3β2, whereas homomeric AChRs are made of five copies of a single α subunit (e.g., α7-α9)65. In contrast, muscle nicotinic AChRs is

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comprised of fetal AChR, which carries the α, β, δ, and γ subunits with a

Each subunit of muscle nicotinic AChR carries an extracellular domain, four

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transmembrane domains (TMD1-4), a short cytoplasmic loop between TMD1 and TMD2, a short extracellular linker between TMD2 and TMD3, a long cytoplasmic

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loop between TMD3 and TMD4, and the C-terminal end. Nicotinic muscle AChR has two binding sites for ACh at the interfaces of the extracellular domains of the α−δ and α−γ/α−ε subunits67, 68. Binding of two ACh molecules to AChR stably open the

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channel pore made TMD2 of five subunits, but binding of a single ACh molecule at either the α−δ or α−γ/α−ε interface is also able to open the TMD2 channel pore for a

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short time69. Only cations pass through the AChR channel pore. Unlike sodium, potassium, or calcium channels, AChRs have no selectivity for cations except for α7AChR, which has 10-20 times higher permeability for Ca2+ than Na+70. The long

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cytoplasmic loop of each subunit is for stabilizing the open state of the AChR channel pore71, 72. The long cytoplasmic loops of AChR subunits are also targets of the agrinLRP4-MuSK signaling. AChR-interacting molecules indicated in Fig. 2 are all targeted to the long cytoplasmic loop.

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the γ subunit with a stoichiometry of α2βδε66.

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stoichiometry of α2βδγ, and adult-type AChR, which carries the ε subunit instead of

2.3. Neural agrin Agrin is an extracellular matrix protein that is released from the motor nerve

terminal. Agrin is enriched at the synaptic basal lamina at the NMJ. Agrin has the Nterminal agrin (NtA) domain, nine follistatin-like (FS) domains, two laminin EGFlike (LE) domains, four EGF-like domains, and three laminin globular (LG) domains, a sperm protein enterokinase and agrin (SEA) domain (Fig. 3A). Three LG domains (LG1, LG2, and LG3) at its C-terminus of agrin are essential and sufficient for inducing MuSK phosphorylation and AChR clustering. The Y site in LG2 is

alternatively spliced (Fig. 3B)73. The Y site is comprised of alternative 12-nucleotide (nt) exon 32 in human. Alternative splicing of exon 32 generates Y0 and Y4 isoforms with 0 and 4 additional amino acids, respectively. Four amino acids in Y4 are required for agrin to interact with heparin. The Y4 isoform is abundant in the neuronal tissues and is slightly expressed in the heart and muscle in rat. Similarly, the Z site in LG3 is comprised of alternative 24-nt exon 36 and 33-nt exon 37 in human (Fig. 3B). Alternative splicing of exons 36 and 37 generates Z0, Z8, Z11 and Z19 isoforms with

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0, 8, 11, and 19 additional amino acids, respectively. Z8, Z11, and Z19 isoforms

induce AChR clustering and the NMJ formation during development. Z8 and Z19

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AChR clustering. Alternative splicing at the Y and Z sites are cooperatively regulated.

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When the 12-nt exon 32 at the Y site is skipped, the exons 36 and 37 at the Z site are always skipped73. Thus, inclusion of the 12-nt exon 32 at the Y site (Y4) is required to

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make Z8, Z11, and Z19 isoforms. The Y0Z0 isoform lacking exons 32, 36, and 37 at both Y and Z sites loses the ability to induce AChR clustering and is expressed in non-neuronal tissues, whereas the Y4Z8, Y4Z11 and Y4Z19 isoforms, which are

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expressed in neuronal tissues, have different AChR-clustering activities. RNAbinding proteins, Nova1 and Nova2, promote inclusion of the 24-nt exon 36 and

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generate Z8 isoform74. Enhancement of inclusion of the 24-nt exon 36 at the Z site of human AGRN to generate Y4Z8 or Y4Z19 isoforms may be a therapeutic target to enhance AChR clustering. Increasing the expression and secretion levels of neural

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agrin will also become a therapeutic target for defective NMJ signal transmission. Two CMS patients with AGRN mutations have been reported75, 76. Both

p.G1709R75 and p.V1727F76 mutations are in the LG2 domain, and are upstream of the neuron-specific Y and Z sites. A nonsense mutation, p.Q353X, is present on the allele in the second patient76. In cultured myotubes, p.G1709R had no effect on

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isoforms carrying exon 36 are more potent than Z11 isoform carrying exon 37 for

MuSK activation or agrin binding to α-dystroglycan. Forced expression of the mutant

mini-agrin in the mouse soleus muscle, however, induced changes similar to those at patient endplates75. In contrast, p.V1727F strikingly reduced MuSK phosphorylation and AChR clustering76. Autoantibodies against agrin are reported in MG patients by three independent groups10, 11, 77. Most of these patients, however, carry autoantibodies against either AChR, MuSK, or LRP4. Neither passive transfer of the patient’s IgG to small animals nor active immunization of small animals with agrin has been performed yet.

Additionally, autoantibodies against agrin are reported in 13.8% of ALS patients78. Thus, pathogenicity of autoantibodies against agrin remains to be elucidated.

2.4. LRP4 Agrin does not directly bind to MuSK79, but binds to LRP4, a member of the low-density lipoprotein (LDL) receptor (LDLR) family, to activate MuSK80, 81. Both MuSK and LRP4 are transmembrane proteins and make a heteromeric tetramer. LRP4

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carries eight LDLR domain class A, four epidermal growth factor (EGF)-like domains, a calcium-binding EGF-like domain, four domains of LDLR class B repeats (β-

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Each stretch of LDLR class B repeats contains five tandem repeats of a YWTD motif

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to build a propeller-like structure. NPSY close to the C-terminal end is a motif for endocytosis and ESQV at the C-terminal end is a motif for binding to PDZ-containing

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proteins. The fourth and fifth LDLR type A repeats, as well as the third β-propeller domain, of LRP4 associate with MuSK (Fig. 1)82. Enhanced binding of LRP4 to kinase domain.

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MuSK by agrin triggers phosphorylation and activation of the MuSK intracellular LRP4 is also expressed at the motor nerve terminal and provides a retrograde

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signal for presynaptic differentiation at the NMJ83, 84. Its ligand(s) and the downstream signaling pathway(s), however, remain to be elucidated. In addition to its specific AChR clustering-inducing role at the NMJ, LRP4 is

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also a well-characterized inhibitor of the Wnt signaling pathway. As a Wnt inhibitor, LRP4 is involved in the formation of skeleton and kidney. Mutations in LRP4 have been reported in Cenani-Lenz syndactyly syndrome (CLSS)85, sclerosteosis-2

(SOST2)86, and low bone mineral density in human87 and mice88. Similarly, Lrp4 mutations cause mule foot disease in cow89, and kidney and limb defects in mouse90.

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propeller domains), a transmembrane domain, and an intracellular domain (Fig. 4).

In addition, a missense SNP rs2306029 in LRP4 is associated with 4.17-fold increase in the risk of developing Richter syndrome91. We reported in two CMS patients that missense mutations in the third βpropeller domain impair binding of LRP4 to agrin and MuSK, and subsequent phosphorylation of MuSK92, 93. In contrast to mutations causing skeletal and kidney diseases stated above, the CMS mutations had no effect on the suppressive effect of LRP4 on the Wnt signaling pathway. Conversely, we confirmed that two missense

mutations in the third β-propeller domain causing SOST2 compromised the suppressive effect of LRP4 on the Wnt signaling pathway, but had no effect on binding of LRP4 to MuSK or agrin. Analysis of additional artificial mutations disclosed that the edge of the third β-propeller domain mediates the MuSK signaling, whereas its central cavity mediates the Wnt signaling. Autoantibodies against LRP4 are reported in patients with MG7, 9, 77. Similar to

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autoantibodies against agrin, about a half of patients with LRP4 antibodies carry autoantibodies against either AChR, MuSK, or agrin. Neither passive transfer of the

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agrin, autoantibodies against LRP4 are reported in 9.8% of ALS patients78. As the specificity of the cell-based assays used to detect anti-LRP4 antibodies, as well as

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anti-agrin antibodies, is variable, it is difficult to be confident of the actual numbers of individuals who are truly positive for harboring autoantibodies. We thus need to

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validate the pathogenicity of autoantibodies against LRP4, as well as agrin.

2.5. MuSK

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MuSK is an indirect receptor for agrin, but its kinase domain is an essential effector of agrin94, 95. As stated above, MuSK binds to LRP4 to receive agrin

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signaling. We reported that agrin enhances interaction between LRP4 and MuSK 36fold in vitro6. A missense mutation in the Ig1 domain of MuSK prevents binding to

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LRP4 and attenuates agrin-stimulated MuSK phosphorylation82. We also dissected MuSK domains and found that immunoglobulin-like (Ig) domains 1 and 4 of MuSK are binding partners that associate with LRP46. Thus, two domains of MuSK associate with two domains of LRP4 (Fig. 1), although the mutual interactions between these domains remain to be elucidated. In addition to binding of MuSK to LRP4, MuSK also binds to collagen Q

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patient’s IgG nor active immunization of LRP4 has been reported. Again, similar to

(ColQ)96 and biglycan97, both of which are extracellular molecules (Fig. 1). At the NMJ, three tetramers of acetylcholinesterase (AChE) are linked to the triple helical ColQ98, 99. AChE/ColQ complex is anchored to the synaptic basal lamina by two mechanisms. First, a pair of heparan sulfate proteoglycan-binding domains (HSPBDs) in the collagen domain of ColQ bind to heparan sulfate proteoglycans including perlecan100-102. Second, the C-terminal domain (CTD) of ColQ binds to MuSK96. We and others have reported that mutations at CTD in CMS compromise anchoring of

AChE/ColQ complex to the NMJ96, 102, 103. We also showed that the HSPBDs and CTD of ColQ can be exploited for the protein-anchoring therapy of ColQ for Colqknockout mice104. In Colq-/- mice, membrane-bound MuSK is reduced in myotubes105, which likely accounts for attenuated clustering of AChRs in Colqknockout mice106. MuSK also binds to a small leucine-rich proteoglycan, biglycan97, 107

, but the functional significance of biglycan on the postsynaptic membrane remains

unsolved.

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The ectodomain of MuSK has three immunoglobulin (Ig)-like domains (Ig1, Ig2, and Ig3) and a frizzled-like cysteine-rich domain (Fz-CRD) (Fig. 1)108-110. Fz-

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(Ig4) containing four cysteines94, 111. Frizzled proteins are receptors for Wnt ligands

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and have ten highly conserved cysteine residues forming five disulfide bonds112. The crystal structure of MuSK Fz-CRD is similar to those of Fizzled proteins110. Deletion

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of Fz-CRD of MuSK in mice may60 or may not61 cause a defect in embryonic prepatterning of AChR clusters. RNA-binding proteins, hnRNP C, YB-1, and hnRNP L coordinately enhance skipping of human MUSK exon 10 encoding the C6 box to

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generate a Wnt-insensitive MuSK isoform113. The C6-deficient MuSK isoform is elusive.

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unique to humans and is not present in mouse, but its functional significance remains MUSK mutations have been reported in CMS patients. Four missense mutations have been functionally characterized, and all MUSK mutations affect agrin-

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mediated AChR clustering either by compromising interaction with Dok-7 and/or LRP4114-116. Two additional missense and two frameshifting mutations have also been reported in MUSK but without functional characterizations117, 118. Autoantibodies against MuSK were first reported in patients with MG who

were negative for anti-AChR antibodies in 20014. Passive transfer of patients’ serum

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CRD is comprised of the C6 box carrying six cysteines and the fourth Ig-like domain

or IgG to mice simulated MG119-122. Similarly, active immunization of mice and

rabbits with MuSK recapitulated MG123-125. Being prompted by observations that cholinesterase inhibitors are not effective for MuSK-MG patients, we showed that anti-MuSK-IgG in MG patients blocks binding of MuSK and ColQ126. We6 and others127, 128 later found that anti-MuSK-IgG blocks binding of MuSK and LRP in the presence of agrin, but not in the absence of agrin. We also showed that inhibition of binding of MuSK to LRP4, and not to ColQ, causes AChR deficiency in MuSK MG

by injecting anti-MuSK-IgG isolated from MuSK-MG patients to Colq-knockout mice6.

2.6 Dok-7 Dok-7 is one of seven Dok family proteins, which carry the pleckstrinhomology (PH) and PTB domains in the N-terminal portion and Src homology 2 (SH2) domain target motifs in the C-terminal region to recruit Crk and Crk-L 26, 129.

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Being prompted by an observation that Dok-7 is highly expressed at the motor

endplate, Okada and colleagues reported that Dok-7 is a non-catalytic adaptor protein

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are addressed in Section 2.1, and are not repeated here.

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Mutations in DOK7 were reported in 21 cases with limb-girdle type CMS with endplate AChR deficiency130. No tubular aggregates in the skeletal muscle have been

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documented in DOK7-CMS patients131. As of Aug. 2017, 79 pathogenic and 2 likely pathogenic DOK7 mutations are registered in ClinVar, a database of clinically relevant variants132. DOK7 is thus one of the major genes causing CMS after CHRNE

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encoding the acetylcholine receptor ε subunit. DOK7 mutations exert their pathogenic effects by compromising Dok-7 expression133 and/or MuSK activation129.

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Interestingly, cholinesterase inhibitors worsen muscle weakness131, 134, although its underlying mechanisms remain elusive. Adrenergic stimulants, ephedrine135-137 and

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albuterol137, 138, ameliorate muscle weakness in DOK7-CMS.

3. Expert opinion

Defective NMJ signal transduction is observed in a number of autoimmune,

hereditary, and sporadic diseases, as well as in intoxication by natural and artificial compounds (Fig. 1). Most of the diseases are orphan diseases except for sarcopenia,

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required for AChR clustering at the motor endplate25. Physiological roles of Dok-7

which is a common malady affecting more than 40% of elderly people at ages 70 years or more18. Approximately 50 million people are currently affected by sarcopenia worldwide, and the number is predicted to increase 10 times in the year 2050139. No drug is currently available for sarcopenia. Considering a role of the defective NMJ signal transduction in sarcopenia17-19, amelioration of the signal transduction at the NMJ is one of the promising targets for drug development. Cholinesterase inhibitors are the most commonly used compounds in clinical practice to increase the number of ACh molecules at the synaptic space and to

enhance opening of AChRs at the motor endplate for facilitation of NMJ signal transduction. Another compound is 3,4-diaminopyridine (3,4-DAP), which blocks the presynaptic voltage-gated potassium channel (VGKC) to slow the repolarization of the action potential delivered to the nerve terminal140. Although 3,4-DAP is effective especially for Lambert-Eaton myasthenic syndrome140 and most forms of CMS141, 3,4-DAP is not an approved drug in many countries. In addition, 3,4-DAP has a frequent adverse effect of paresthesia (8.8%), and rare adverse effects of sleep study of 669 patients with multiple sclerosis taking 3,4-DAP142.

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disturbances (1.2%) and nausea/vomiting (0.9%), according to a retrospective cohort

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many human genes. Neuron-specific splicing at Y and Z sites of AGRN generates

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neural agrin, which is capable of inducing AChR clustering (Fig. 3B). Although the splicing mechanisms of AGRN have not been dissected except for the roles of Nova1

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and Nova274, enhancement of alternative splicing at Y and Z sites may be a promising therapeutic target for defective NMJ signal transduction. Similarly, human MUSK exon 10 is alternatively skipped by coordinated actions of hnRNP C, YB-1, and

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hnRNP L to generate a Wnt-insensitive MuSK isoform113. Suppression of the actions of these RNA-binding proteins can also be a therapeutic target. Although not

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addressed in the text above, we are currently dissecting alternative splicing mechanisms of AGRN, DOK7, and GFPT1. Modulation of alternative splicing of these genes may also enhance the NMJ signal transduction.

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LRP4 and MuSK have extracellular domains, which can be potentially

activated by membrane-impermeable compounds or antibodies. In addition, the agrinLRP4-MuSK signaling pathway involves a lot of kinases and possibly phosphatases. Modulation of kinase and phosphatase activities, as well as overexpression of catalytic and non-catalytic adaptor proteins, will also be promising therapeutic options

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Genes encoding the NMJ molecules are alternatively spliced, as observed in

to enhance the NMJ signal transduction. In addition to agrin, Wnt ligands and Rspo2 are able to induce MuSK-mediated AChR clustering. Induction of Wnt ligands20 and

Rspo263, as well as modulation of their signaling molecules, will become attractive therapeutic strategies.

Acknowledgements

We would like to acknowledge Keiko Itano at Nagoya University Graduate School of Medicine for finalizing figures.

Funding Work in the authors’ laboratory are supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labour and Welfare; Japan Agency for Medical Research and Development, and the Smoking

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Declaration of Interest

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The authors have no relevant affiliations or financial involvement with any

organization or entity with a financial interest in or financial conflict with the subject

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matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or

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patents received or pending, or royalties.

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Research Foundation.

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142.

Figu ure Legend ds Fig.. 1. Molecuular architecture of the N NMJ. Doub ble headed arrows a pointt to interactting dom mains. NMJ molecules and their asssociated hu uman diseasses are indiccated in blacck and red letters, respectivelly. Moleculees causing MG M and CM MS are not iindicated. MG M 7 is caaused by auutoantibodiees against A AChR, MuSK K4-6, LRP47-9 , and agriin10, 11. Anti-

MuS SK antibodyy (not show wn) and ColQ Q competitively bind to Ig1 and Igg4 of MuSK K.

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Antti-MuSK anntibody thus displaces C ColQ from MuSK. M Both h anti-MuS K antibody y and ColQ suppress MuSK-LRP P4 interactiion, but the suppressivee effect of aanti-MuSK

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1 14 encooding NMJ molecules13, . Amonng the indicaated NMJ molecules, m C CMS mutatio ons

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have been repoorted in genees encodingg AChR sub bunits, rapsy yn, agrin, LR RP4, MuSK K, Dokk-7, ColQ, voltage-gate v ed sodium cchannel, and d SNAP-25. LDLR-A iin LPR4, lo ow-

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density lipoprootein recepto or type A reepeats. BPD D1-4 in LRP P4, the first tto fourth β-proppeller domaains. Ig1-4 in MuSK, im mmunoglob bulin-like do omains 1-4. C6 box in MuS SK, a domaain with six cysteines. F Fz-CRD in MuSK, Frizzzled-like cy cysteine-rich h

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dom main compriised of C6 box b and Ig44 domain.

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antiibody is more than thatt of ColQ. C CMS is caussed by germ mline mutatioons in 25 geenes

Fig.. 2. Intracellular signaling molecul ules involved d in MuSK phosphoryllation and AChhR clusterinng. The cyto oplasmic doomain of MuSK and the long cytopplasmic loo ops of A AChR subunnits are enlaarged to inddicate interactions with intracellulaar signaling mollecules. Redd arrows ind dicate kinas e activities.. P represen nts phosphorrylation.

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hum man agrin geene (B). (A)) A 95-kDa neural agriin fragment (not shownn) downstreaam

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to E EG1 is sufficcient to indu uce AChR cclustering. (B) ( Alternattive splicingg of at the Y and Z sites in thhe LG2 and d LG3 domaains, respectively, geneerates neuraal agrin

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isofforms (Y4Z8, Y4Z11, and a Y4Z19)), which aree capable off inducing A AChR

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Fig.. 3. Domainn structure of o agrin (A) and alternaative splicin ng at the Y aand Z sites of o

Fig.. 4. Domainn structure of o LRP4. Thhe fourth an nd fifth LDL LR class A rrepeats and the thirdd β-propelleer domain, which w is coomprised of the third strretch of LD DLR class B repeeats, bind too MuSK82.

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