Adeno-Associated Virus Type 9-Mediated Gene Therapy of Choline Acetyltransferase-Deficient Mice.

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene (CHAT) result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for Chat (Chat-/-) die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing loxP sites flanking Chat exons 4 and 5 with mice that expressed Cre-ERT2. Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of Chat, autonomic failure, weakness, and death. However, a proportion of Chatflox/flox-Cre-ERT2 mice receiving at birth an intracerebroventricular injection of 2 × 1013 vg/kg adeno-associated virus type 9 (AAV9) carrying human CHAT (AAV9-CHAT) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9-CHAT by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of Chatflox/flox-Cre-ERT2 mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human CHAT RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CHAT-CMS.

Intravenous immunoglobulin is safe and effective in controlling pre-existing paraneoplastic neuromuscular diseases in cancer patients treated with immune checkpoint inhibitors: two case reports and literature review.

Immune checkpoint inhibitors cause rare but potentially fatal neuromuscular complications, leading to a concern to use these agents in cancer patients with pre-existing autoimmune or inflammatory neuromuscular diseases. We report two such patients with paraneoplastic dermatomyositis and "seronegative" paraneoplastic demyelinating neuropathy, respectively, who have been successfully treated with immune checkpoint inhibitor monotherapy as well as maintenance intravenous immunoglobulin. While controlling the paraneoplastic or autoimmune neuromuscular diseases, the use of intravenous immunoglobulin did not compromise the anti-cancer effect of immune checkpoint inhibitor.

Dominant and recessive congenital myasthenic syndromes caused by SYT2 mutations.

INTRODUCTION/AIMS: We studied a patient with a congenital myasthenic syndrome (CMS) caused by a dominant mutation in the synaptotagmin 2 gene (SYT2) and compared the clinical features of this patient with those of a previously described patient with a recessive mutation in the same gene. METHODS: We performed electrodiagnostic (EDX) studies, genetic studies, muscle biopsy, microelectrode recordings and electron microscopy (EM). RESULTS: Both patients presented with muscle weakness and bulbar deficits, which were worse in the recessive form. EDX studies showed presynaptic failure, which was more prominent in the recessive form. Microelectrode studies in the dominant form showed a marked reduction of the quantal content, which increased linearly with higher frequencies of nerve stimulation. The MEPP frequencies were normal at rest but increased markedly with higher frequencies of nerve stimulation. The EM demonstrated overdeveloped postsynaptic folding, and abundant endosomes, multivesicular bodies and degenerative lamellar bodies inside small nerve terminals. DISCUSSION: The recessive form of CMS caused by a SYT2 mutation showed far more severe clinical manifestations than the dominant form. The pathogenesis of the dominant form likely involves a dominant-negative effect due to disruption of the dual function of synaptotagmin as a Ca2+ -sensor and modulator of synaptic vesicle exocytosis.

Recessive congenital myasthenic syndrome caused by a homozygous mutation in SYT2 altering a highly conserved C-terminal amino acid sequence.

Defects in the gene encoding synaptotagmin 2 (SYT2) have been linked to a presynaptic congenital myasthenic syndrome (CMS) and motor neuropathies. However, to date only dominant forms of the disease have been described. We report here a consanguineous patient with a severe recessive form of presynaptic CMS and denervation atrophy caused by the homozygous mutation c.1191delG, p.Arg397Serfs*37 in SYT2. The affected 2-year-old girl had profound weakness and areflexia with moderate bulbar deficit. Repetitive nerve stimulation revealed an extreme reduction of compound muscle action potential amplitudes at rest, with a striking facilitation followed by a progressive decline at fast stimulation rates. These findings were reminiscent, but not identical to those seen in the Lambert-Eaton myasthenic syndrome. 3,4 diaminopyridine and pyridostigmine were effective to ameliorate muscle fatigue, but albuterol was ineffective. Modeling of the mutation using the rat Syt1 C2B x-ray structure revealed that Arg397Serfs*37 disrupts a highly conserved amino acid sequence at the bottom face of the C2B domain not directly involved in calcium binding, but crucial for synaptotagmin-SNARE interaction and exocytosis. Thus, this report describes a recessive form of synaptotagmin 2-CMS and highlights the importance of the synaptotagmin C-terminal on synaptic vesicle fusion and exocytosis.

Phenotypic Differences in 2 Unrelated Cases Carrying Identical DOK7 Mutations.

INTRODUCTION: Mutations in the Dok-7 gene (DOK7) underlie a congenital myasthenic syndrome (CMS) with a characteristic limb-girdle (LG) pattern of muscle weakness. Multiple clinical findings and a wide clinical heterogeneity have been identified in this form of CMS. METHODS: We describe here 2 unrelated adult patients who presented with a LG CMS, caused by 2 compound heterozygous pathogenic sequence variants in DOK7: c.1124_1127dupTGCC (P.Ala378Serfs*30) and c.480C> A (p.Tyr160*). RESULTS: Although both patients presented with severe proximal weakness consistent with LG myasthenia, one of the patients presented with additional distal muscle involvement in the lower extremities. By contrast, the other patient had severe bulbar and respiratory deficit requiring gastric tube feeding and mechanical ventilatory support for most parts of the day. DISCUSSION: These 2 cases illustrate the lack of phenotype-genotype correlation and the absence of geographic, genetic, and ethnic association in cases of LG CMS caused by DOK7 mutations.

Pathogenic effects of agrin V1727F mutation are isoform specific and decrease its expression and affinity for HSPGs and LRP4.

Agrin is a large extracellular matrix protein whose isoforms differ in their tissue distribution and function. Motoneuron-derived y+z+ agrin regulates the formation of the neuromuscular junction (NMJ), while y-z- agrin is widely expressed and has diverse functions. Previously we identified a missense mutation (V1727F) in the second laminin globular (LG2) domain of agrin that causes severe congenital myasthenic syndrome. Here, we define pathogenic effects of the agrin V1727F mutation that account for the profound dysfunction of the NMJ. First, by expressing agrin variants in heterologous cells, we show that the V1727F mutation reduces the secretion of y+z+ agrin compared to wild type, whereas it has no effect on the secretion of y-z- agrin. Second, we find that the V1727F mutation significantly impairs binding of y+z+ agrin to both heparin and the low-density lipoprotein receptor-related protein 4 (LRP4) coreceptor. Third, molecular modeling of the LG2 domain suggests that the V1727F mutation primarily disrupts the y splice insert, and consistent with this we find that it partially occludes the contribution of the y splice insert to agrin binding to heparin and LRP4. Together, these findings identify several pathogenic effects of the V1727F mutation that reduce its expression and ability to bind heparan sulfate proteoglycan and LRP4 coreceptors involved in the muscle-specific kinase signaling pathway. These defects primarily impair the function of neural y+z+ agrin and combine to cause a severe CMS phenotype, whereas y-z- agrin function in other tissues appears preserved.

Presynaptic congenital myasthenic syndrome with altered synaptic vesicle homeostasis linked to compound heterozygous sequence variants in RPH3A.

BACKGROUND: Monogenic defects of synaptic vesicle (SV) homeostasis have been implicated in many neurologic diseases, including autism, epilepsy, and movement disorders. In addition, abnormal vesicle exocytosis has been associated with several endocrine dysfunctions. METHODS: We report an 11 year old girl with learning disabilities, tremors, ataxia, transient hyperglycemia, and muscle fatigability responsive to albuterol sulfate. Failure of neuromuscular transmission was confirmed by single fiber electromyography. Electron microscopy of motor nerve terminals revealed marked reduction in SV density, double-membrane-bound sacs containing SVs, abundant endosomes, and degenerative lamellar bodies. The patient underwent whole exome sequencing (WES) and relevant sequence variants were expressed and studied in a mammalian cell line. RESULTS: Chromosomal microarray studies and next generation sequencing (NGS) of mitochondrial DNA were unrevealing; however, NGS of genomic DNA showed two rare sequence variants in the gene encoding rabphilin 3a (RPH3A). The paternally inherited variant c.806 G>A (p.Arg269Gln) involves a substitution of a conserved residue in the linker region, while the maternally inherited variant c.1390 G>T (p.Val464Leu) involves a conserved amino acid substitution in the highly conserved C2A region. Expression studies revealed that p.Arg269Gln strongly impairs the binding of rabphilin 3a to 14-3-3, which is a proposed regulator of synaptic transmission and plasticity. In contrast, the binding of rabphilin 3a to 14-3-3 is only marginally impaired by p.Val464Leu; thus, the pathogenic role of p.Val464Leu remains unclear. CONCLUSION: In summary, we report a patient with a multisystem neurologic disorder and altered SV regulation attributed to defects in RPH3A, which grants further studies of this gene in human disorders of synaptic transmission.

A presynaptic congenital myasthenic syndrome attributed to a homozygous sequence variant in LAMA5.

We report a severe defect of neuromuscular transmission in a consanguineous patient with a homozygous variant in the laminin α5 subunit gene (LAMA5). The variant c.8046C > T (p.Arg2659Trp) is rare and has a predicted deleterious effect. The affected individual, who also carries a rare homozygous sequence variant in LAMA1, had normal cognitive function, but magnetic resonance brain imaging showed mild volume loss and periventricular T2 prolongation. Repetitive nerve stimulation at 2 Hz showed 50% decrement of compound muscle action potential amplitudes but 250% facilitation immediately after exercise, similar to that seen in Lambert-Eaton myasthenic syndrome. Endplate studies demonstrated a profound reduction of the endplate potential quantal content but normal amplitudes of miniature endplate potentials. Electron microscopy showed endplates with increased postsynaptic folding that were denuded or only partially occupied by small nerve terminals. Expression studies revealed that p.Arg2659Trp caused decreased binding of laminin α5 to SV2A and impaired laminin-521 cell adhesion and cell projection support in primary neuronal cultures. In summary, this report describing severe neuromuscular transmission failure in a patient with a LAMA5 mutation expands the list of phenotypes associated with defects in genes encoding α-laminins.

Congenital Myasthenic Syndrome due to DOK7 mutations in a family from Chile.

Congenital myasthenic syndromes (CMS) are neuromuscular transmission disorders caused by mutations in genes encoding neuromuscular junction proteins. A 61-year-old female and her older sister showed bilateral ptosis, facial and proximal limb weakness, and scoliosis since childhood. Another female sibling had milder signs, while other family members were asymptomatic. Facial nerve repetitive stimulation in the proband showed decrement of muscle responses. Single fiber EMG revealed increased jitter and blocking. Muscle biopsy showed type 2-fiber atrophy, without tubular aggregates. Mutational analysis in the three affected siblings revealed two compound heterozygous mutations in DOK7: c.1457delC, that predicts p.Pro486Argfs*13 and truncates the protein C-terminal domain, and c.473G>A, that predicts p.Arg158Gln and disruption of the dok7-MuSK interaction in the phosphotyrosine binding (PTB) domain. Unaffected family members carried only one or neither mutation. Discussion: Two of the affected sisters showed marked improvement with salbutamol treatment, which illustrates the benefits of a correct diagnosis and treatment of DOK7-CMS.

Presynaptic congenital myasthenic syndrome with a homozygous sequence variant in LAMA5 combines myopia, facial tics, and failure of neuromuscular transmission.

Defects in genes encoding the isoforms of the laminin alpha subunit have been linked to various phenotypic manifestations, including brain malformations, muscular dystrophy, ocular defects, cardiomyopathy, and skin abnormalities. We report here a severe defect of neuromuscular transmission in a consanguineous patient with a homozygous variant in the laminin alpha-5 subunit gene (LAMA5). The variant c.8046C>T (p.Arg2659Trp) is rare and has a predicted deleterious effect. The affected individual, who also carries a rare homozygous sequence variant in LAMA1, had muscle weakness, myopia, and facial tics. Magnetic resonance imaging of brain showed mild volume loss and periventricular T2 prolongation. Repetitive nerve stimulation revealed 50% decrement of compound muscle action potential amplitudes and 250% facilitation immediately after exercise, Endplate studies identified a profound reduction of the endplate potential quantal content and endplates with normal postsynaptic folding that were denuded or partially occupied by small nerve terminals. Expression studies revealed that p.Arg2659Trp caused decreased binding of laminin alpha-5 to SV2A and impaired laminin-521 cell-adhesion and cell projection support in primary neuronal cultures. In summary, this report describing severe neuromuscular transmission failure in a patient with a LAMA5 mutation expands the list of phenotypes associated with defects in genes encoding alpha-laminins.

Effect of 3,4-diaminopyridine at the murine neuromuscular junction.

INTRODUCTION: We investigated the effects of 3,4-diaminopyridine (3,4-DAP) and its acetylated metabolite, N-(4-amino-pyridin-3-yl) acetamide (3-Ac), at the mammalian neuromuscular junction. METHODS: Quantal release of acetylcholine was studied in diaphragm muscles of mice, using in vitro intracellular microelectrode recordings. RESULTS: Under conditions of low probability of release, 3,4-DAP produced a 1,000% increase in quantal release, but 3-Ac had no effect. Under conditions of normal probability of release, the effect of 3,4-DAP was modest and limited by concurrent depletion of synaptic vesicles, especially with high concentrations of 3,4-DAP and high frequencies of nerve stimulation. CONCLUSIONS: These findings predict 3,4-DAP is most effective in conditions with low probability of quantal release, such as Lambert-Eaton myasthenic syndrome. A beneficial effect is also expected in disorders of neuromuscular transmission in which the effect of 3,4-DAP on quantal release is not limited by depletion of synaptic vesicles, such as postsynaptic congenital myasthenic syndromes. Muscle Nerve, 2016 Muscle Nerve 55: 223-231, 2017.

Choline Acetyltransferase Mutations Causing Congenital Myasthenic Syndrome: Molecular Findings and Genotype-Phenotype Correlations.

Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype-phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of phosphorylated ChAT of seven CHAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys, and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal stability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp, and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met, which is located far from both active and substrate-binding sites, produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes.

Defective fast inactivation recovery of Nav 1.4 in congenital myasthenic syndrome.

OBJECTIVE: To describe the unique phenotype and genetic findings in a 57-year-old female with a rare form of congenital myasthenic syndrome (CMS) associated with longstanding muscle fatigability, and to investigate the underlying pathophysiology. METHODS: We used whole-cell voltage clamping to compare the biophysical parameters of wild-type and Arg1457His-mutant Nav 1.4. RESULTS: Clinical and neurophysiological evaluation revealed features consistent with CMS. Sequencing of candidate genes indicated no abnormalities. However, analysis of SCN4A, the gene encoding the skeletal muscle sodium channel Nav 1.4, revealed a homozygous mutation predicting an arginine-to-histidine substitution at position 1457 (Arg1457His), which maps to the channel's voltage sensor, specifically D4/S4. Whole-cell patch clamp studies revealed that the mutant required longer hyperpolarization to recover from fast inactivation, which produced a profound use-dependent current attenuation not seen in the wild type. The mutant channel also had a marked hyperpolarizing shift in its voltage dependence of inactivation as well as slowed inactivation kinetics. INTERPRETATION: We conclude that Arg1457His compromises muscle fiber excitability. The mutant fast-inactivates with significantly less depolarization, and it recovers only after extended hyperpolarization. The resulting enhancement in its use dependence reduces channel availability, which explains the patient's muscle fatigability. Arg1457His offers molecular insight into a rare form of CMS precipitated by sodium channel inactivation defects. Given this channel's involvement in other muscle disorders such as paramyotonia congenita and hyperkalemic periodic paralysis, our study exemplifies how variations within the same gene can give rise to multiple distinct dysfunctions and phenotypes, revealing residues important in basic channel function.

Axonal neuropathy in female carriers of the fragile X premutation with fragile x-associated tremor ataxia syndrome.

INTRODUCTION: In this study we examined whether females with the fragile X-associated tremor ataxia syndrome (FXTAS) and non-FXTAS premutation carriers have electrophysiological signs of underlying peripheral neuropathy. METHODS: Nerve conduction studies (NCS) were performed on 19 women with FXTAS, 20 non-FXTAS carriers, and 26 age-matched controls. The results were compared with existing data on corresponding male carriers. RESULTS: Women with FXTAS and non-FXTAS carriers had reduced sensory nerve action potential amplitudes. Also, there was a strong trend for reduced compound muscle action potential amplitudes in women with FXTAS, but not in non-FXTAS carriers. No significant slowing of nerve conduction velocities, prolongation of F-wave latencies, or associations with molecular measures was observed. CONCLUSIONS: This study suggests an underlying axonal neuropathy in women with FXTAS. However, in comparison to men with FXTAS, the NCS abnormalities in women were less severe, possibly due to the effect of a normal X chromosome.

COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina.

Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.

Animal models of antimuscle-specific kinase myasthenia.

Antimuscle-specific kinase (anti-MuSK) myasthenia (AMM) differs from antiacetylcholine receptor myasthenia gravis in exhibiting more focal muscle involvement (neck, shoulder, facial, and bulbar muscles) with wasting of the involved, primarily axial, muscles. AMM is not associated with thymic hyperplasia and responds poorly to anticholinesterase treatment. Animal models of AMM have been induced in rabbits, mice, and rats by immunization with purified xenogeneic MuSK ectodomain, and by passive transfer of large quantities of purified serum IgG from AMM patients into mice. The models have confirmed the pathogenic role of the MuSK antibodies in AMM and have demonstrated the involvement of both the presynaptic and postsynaptic components of the neuromuscular junction. The observations in this human disease and its animal models demonstrate the role of MuSK not only in the formation of this synapse but also in its maintenance.

Dominantly inherited myotonia congenita resulting from a mutation that increases open probability of the muscle chloride channel CLC-1.

Myotonia congenita-inducing mutations in the muscle chloride channel CLC-1 normally result in reduced open probability (P (o)) of this channel. One well-accepted mechanism of the dominant inheritance of this disease involves a dominant-negative effect of the mutation on the function of the common-gate of this homodimeric, double-barreled molecule. We report here a family with myotonia congenita characterized by muscle stiffness and clinical and electrophysiologic myotonic phenomena transmitted in an autosomal dominant pattern. DNA sequencing of DMPK and ZNF9 genes for myotonic muscular dystrophy types I and II was normal, whereas sequencing of CLC-1 encoding gene, CLCN1, identified a single heterozygous missense mutation, G233S. Patch-clamp analyses of this mutant CLC-1 channel in Xenopus oocytes revealed an increased P (o) of the channel's fast-gate, from ~0.4 in the wild type to >0.9 in the mutant at -90 mV. In contrast, the mutant exhibits a minimal effect on the P (o) of the common-gate. These results are consistent with the structural prediction that the mutation site is adjacent to the fast-gate of the channel. Overall, the mutant could lead to a significantly reduced dynamic response of CLC-1 to membrane depolarization, from a fivefold increase in chloride conductance in the wild type to a twofold increase in the mutant-this might result in slower membrane repolarization during an action potential. Since expression levels of the mutant and wild-type subunits in artificial model cell systems were unable to explain the disease symptoms, the mechanism leading to dominant inheritance in this family remains to be determined.

Synaptic basal lamina-associated congenital myasthenic syndromes.

Proteins associated with the basal lamina (BL) participate in complex signal transduction processes that are essential for the development and maintenance of the neuromuscular junction (NMJ). Most important junctional BL proteins are collagens, such as collagen IV (α3-6), collagen XIII, and ColQ; laminins; nidogens; and heparan sulfate proteoglycans, such as perlecan and agrin. Mice lacking Colq (Colq(-/-)), laminin ß2 (Lamb2(-/-)), or collagen XIII (Col13a1(-/-)) show immature nerve terminals enwrapped by Schwann cell projections that invaginate into the synaptic cleft and decrease contact surface for neurotransmission. Human mutations in COLQ, LAMB2, and AGRN cause congenital myasthenic syndromes (CMSs) owing to deficiency of ColQ, laminin-ß2, and agrin, respectively. In these syndromes the NMJ ultrastructure shows striking resemblance to that of mice lacking the corresponding protein; furthermore, the extracellular localization of mutant proteins may provide favorable conditions for replacement strategies based on gene therapy and stem cells.

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.