Neuromuscular Junction Defects Research Articles

HuD impairs neuromuscular junctions and induces apoptosis in human iPSC and Drosophila ALS models

Defects at the neuromuscular junction (NMJ) are among the earliest hallmarks of amyotrophic lateral sclerosis (ALS). According to the “dying-back” hypothesis, NMJ disruption not only precedes but also triggers the subsequent degeneration of motoneurons in both sporadic (sALS) and familial (fALS) ALS. Using human induced pluripotent stem cells (iPSCs), we show that the RNA-binding protein HuD (ELAVL4) contributes to NMJ defects and apoptosis in FUS-ALS. HuD overexpression mimics the severe FUSP525L mutation, while its knockdown rescues the FUSP525L phenotypes. In Drosophila, neuronal overexpression of the HuD ortholog, elav, induces motor dysfunction, and its knockdown improves motor function in a FUS-ALS model. Finally, we report increased HuD levels upon oxidative stress in human motoneurons and in sALS patients with an oxidative stress signature. Based on these findings, we propose that HuD plays a role downstream of FUS mutations in fALS and in sALS related to oxidative stress.

Mitochondrial disorders are associated with morphological neuromuscular junction defects

We aimed to evaluate whether inherited mitochondrial dysfunction is associated with neuromuscular junction remodeling in patients with mitochondrial disorders.Muscle biopsies from 15 patients with mitochondrial disorders and 10 control patients were analyzed through immunostaining for various neuromuscular junction components. The patient group, with a mean age of 49.9 years, exhibited various mitochondrial disorders including chronic progressive external ophthalmoplegia, Kearns-Sayre syndrome, and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes. Patients with mitochondrial disorders had a high percentage of remodeled (p= 0.0001), neoformed (p= 0.0049) and dilated (p= 0.016) endplates. There was a trend toward an increased proportion of neuromuscular junctions with terminal Schwann cell extension in these patients (p= 0.052). No significant difference was found in myofiber diameter between the groups. The observed neuromuscular junction defects varied widely across different mitochondrial disorder phenotypes and were present even without accompanying muscle weakness or neuropathy.This suggest that mitochondrial disorders are associated with a primary NMJ remodeling independent of muscle structural damage. Pathomechanisms underpinning this remodeling of the neuromuscular junction, as well as clinical factors predictive of this remodeling, remain to be fully characterized.

Clinical and genetic characterisation of a large Indian congenital myasthenic syndrome cohort.

Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and β2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date. Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014-19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1-56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0-49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES, TEFM) in our cohort accounted for 2.8%. Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected. This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1, GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES, presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects.

Medium-Chain Fatty Acids Rescue Motor Function and Neuromuscular Junction Degeneration in a Drosophila Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterised by progressive degeneration of the motor neurones. An expanded GGGGCC (G4C2) hexanucleotide repeat in C9orf72 is the most common genetic cause of ALS and frontotemporal dementia (FTD); therefore, the resulting disease is known as C9ALS/FTD. Here, we employ a Drosophila melanogaster model of C9ALS/FTD (C9 model) to investigate a role for specific medium-chain fatty acids (MCFAs) in reversing pathogenic outcomes. Drosophila larvae overexpressing the ALS-associated dipeptide repeats (DPRs) in the nervous system exhibit reduced motor function and neuromuscular junction (NMJ) defects. We show that two MCFAs, nonanoic acid (NA) and 4-methyloctanoic acid (4-MOA), can ameliorate impaired motor function in C9 larvae and improve NMJ degeneration, although their mechanisms of action are not identical. NA modified postsynaptic glutamate receptor density, whereas 4-MOA restored defects in the presynaptic vesicular release. We also demonstrate the effects of NA and 4-MOA on metabolism in C9 larvae and implicate various metabolic pathways as dysregulated in our ALS model. Our findings pave the way to identifying novel therapeutic targets and potential treatments for ALS.

Mitochondrial Mutations Can Alter Neuromuscular Transmission in Congenital Myasthenic Syndrome and Mitochondrial Disease.

Congenital myasthenic syndromes (CMS) are a group of rare, neuromuscular disorders that usually present in childhood or infancy. While the phenotypic presentation of these disorders is diverse, the unifying feature is a pathomechanism that disrupts neuromuscular transmission. Recently, two mitochondrial genes-SLC25A1 and TEFM-have been reported in patients with suspected CMS, prompting a discussion about the role of mitochondria at the neuromuscular junction (NMJ). Mitochondrial disease and CMS can present with similar symptoms, and potentially one in four patients with mitochondrial myopathy exhibit NMJ defects. This review highlights research indicating the prominent roles of mitochondria at both the pre- and postsynapse, demonstrating the potential for mitochondrial involvement in neuromuscular transmission defects. We propose the establishment of a novel subcategorization for CMS-mitochondrial CMS, due to unifying clinical features and the potential for mitochondrial defects to impede transmission at the pre- and postsynapse. Finally, we highlight the potential of targeting the neuromuscular transmission in mitochondrial disease to improve patient outcomes.

Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2.

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here, we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

Abnormal decrement on high-frequency repetitive nerve stimulation in congenital myasthenic syndrome with GFPT1 mutations and review of literature.

ObjectivesCongenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders characterized by neuromuscular junction defects. Mutations in GFPT1 have been shown to underlie CMS. An increasing number of patients with CMS due to mutations in GFPT1 have been reported. However, a comprehensive review of clinical and genetic analyses of GFPT-related CMS worldwide is lacking, especially, given that the common or hotspot mutations in GFPT1 have not been reported. Here, we described the clinical and genetic findings of three patients with GFPT1 mutations from southwestern China and reviewed the clinical and genetic features of patients with GFPT1-related CMS worldwide.MethodsClinical, laboratory, electrophysiological, myopathological, and genetic analyses of three patients with GFPT1-related CMS from southwestern China were conducted, and a review of previously published or reported cases about congenital myasthenic syndrome with GFPT1 mutations in the PubMed database was made.ResultsThe clinical, laboratory, electrophysiological, and myopathological features by muscle biopsy of three patients with GFPT1-related CMS were consistent with those of previously reported patients with GFPT1 mutations. Additionally, an abnormal decrement in high-frequency RNS was found. Two different homozygous missense mutations (c.331C>T, p.R111C; c.44C>T, p.T15M) were detected by whole-exome sequencing (WES) or targeted neuromuscular disorder gene panels.ConclusionA distinct decremental response to high-frequency RNS was found in three patients with GFPT1-related CMS from southwestern China, which has never been reported thus far. In addition, the location and degree of tubular aggregates (TAs) seemed to be associated with the severity of clinical symptoms and serum creatine kinase levels, further expanding the phenotypic spectrum of GFPT1-related CMS. Lastly, some potential hotspot mutations in GFPT1 have been found in GFPT1-CMS worldwide.

Genetic and clinical evaluation of congenital myasthenic syndromes with long-term follow-up: experience of a tertiary center in Turkey.

Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders affecting the safety factor which required for neuromuscular transmission. Here we reported our experience in children with CMS. We retrospectively collected the data of 18 patients with CMS who were examined in our outpatient clinic between January 2021 and January 2022. The diagnosis of CMS was based on the presence of clinical symptoms such as abnormal fatigability and weakness, absence of autoantibodies against acetylcholine receptor and muscle-specific kinase, electromyographic evidence of neuromuscular junction defect, molecular genetic confirmation, and response to treatment. The most common mutations were in the acetylcholine receptor (CHRNE) gene (8/18) and choline acetyltransferase (ChAT) (2/18) gene. Despite targeted gene sequencing and whole exome sequencing (WES) were underwent, we couldn't detect a genetic mutation in three out of patients. The most commonly determined initial finding was eyelid ptosis, followed by fatigable weakness, and respiratory insufficiency. Although the most commonly used drug was pyridostigmine, we have experienced that caution should be exercised as it may worsen some types of CMS. We reported in detail the phenotypic features of very rare gene mutations associated with CMS and our experience in the treatment of this disease. Although CMS are rare genetic disorder, the prognosis can be very promising with appropriate treatment in most CMS subtypes.

Therapeutic Potential of CKD-504, a Novel Selective Histone Deacetylase 6 Inhibitor, in a Zebrafish Model of Neuromuscular Junction Disorders.

The neuromuscular junction (NMJ), which is a synapse for signal transmission from motor neurons to muscle cells, has emerged as an important region because of its association with several peripheral neuropathies. In particular, mutations in GARS that affect the formation of NMJ result in Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. These disorders are mainly considered to be caused by neuronal axon abnormalities; however, no treatment is currently available. Therefore, in order to determine whether the NMJ could be targeted to treat neurodegenerative disorders, we investigated the NMJ recovery effect of HDAC6 inhibitors, which have been used in the treatment of several peripheral neuropathies. In the present study, we demonstrated that HDAC6 inhibition was sufficient to enhance movement by restoring NMJ impairments observed in a zebrafish disease model. We found that CKD-504, a novel HDAC6 inhibitor, was effective in repairing NMJ defects, suggesting that treatment of neurodegenerative diseases via NMJ targeting is possible.

Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila

Increasing evidence points to the involvement of cell types other than motor neurons in both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. Studies have since focused on evaluating the contribution of glia in SMA. Here, we made use of the Drosophila model to ask whether the survival motor neuron (Smn) protein, the causative factor for SMA, is required selectively in glia. We show that the specific loss of Smn function in glia during development reduced survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology in flies. In contrast, gain rather than loss of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2–8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). We show that glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability but, similar to Smn reduction, had no negative effect on neuromuscular function. Our findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.

Functional loss of ubiquitin-specific protease 14 may lead to a novel distal arthrogryposis phenotype.

Multiple congenital contractures (MCC) comprise a number of rare, non-progressive conditions displaying marked phenotypic and etiologic heterogeneity. A genetic cause can be established in approximately half of the affected individuals, attributed to genetic defects in the formation and functioning of the central and peripheral nervous system, neuromuscular junctions, skeletal muscles, and connective tissue. Ubiquitin-specific protease 14 (USP14) encodes a major proteasome-associated deubiquitinating enzyme with an established dual role as an inhibitor and an activator of proteolysis, maintaining protein homeostasis. Usp14-deficient mice show a phenotype similar to lethal human MCC phenotypes, with callosal anomalies, muscle wasting, and early lethality, attributed to neuromuscular junction defects due to decreased monomeric ubiquitin pool. We describe a new, autosomal recessive MCC phenotype in three fetuses from two different branches of a consanguineous family, presenting with distal arthrogryposis, underdevelopment of the corpus callosum, and dysmorphic facial features. Exome sequencing identified a biallelic 4-bp deletion (c.233_236delTTCC; p.Leu78Glnfs*11, SCV002028347) in USP14, and sequencing of family members showed segregation with the phenotype. RT-qPCR experiment in an unaffected heterozygote revealed that mutant USP14 was expressed, indicating that abnormal transcript escapes nonsense-mediated mRNA decay. We propose that herein described fetuses represent the first human phenotype of USP14 loss, with callosal anomalies and/or cortical malformations, multiple contractures, and recognizable dysmorphic facial features.

Postsynaptic neuromuscular junction defect in a patient with miller fisher syndrome

Miller Fisher syndrome (MFS) is an acute immune-mediated neuropathy. To date only few studies reported a possible overlap of Myasthenia Gravis (MG) and MFS in patients with acute ophthalmoplegia. We report the case of a patient diagnosed as MFS with a coexistent neuromuscular junction defect.

MG53 Preserves Neuromuscular Junction Integrity and Alleviates ALS Disease Progression.

Respiratory failure from progressive respiratory muscle weakness is the most common cause of death in amyotrophic lateral sclerosis (ALS). Defects in neuromuscular junctions (NMJs) and progressive NMJ loss occur at early stages, thus stabilizing and preserving NMJs represents a potential therapeutic strategy to slow ALS disease progression. Here we demonstrate that NMJ damage is repaired by MG53, an intrinsic muscle protein involved in plasma membrane repair. Compromised diaphragm muscle membrane repair and NMJ integrity are early pathological events in ALS. Diaphragm muscles from ALS mouse models show increased susceptibility to injury and intracellular MG53 aggregation, which is also a hallmark of human muscle samples from ALS patients. We show that systemic administration of recombinant human MG53 protein in ALS mice protects against injury to diaphragm muscle, preserves NMJ integrity, and slows ALS disease progression. As MG53 is present in circulation in rodents and humans under physiological conditions, our findings provide proof-of-concept data supporting MG53 as a potentially safe and effective therapy to mitigate ALS progression.

CONGENITAL MYOPATHIES – NEMALINE MYOPATHIES: EP.40 Neuromuscular junction defects in ACTA1-related nemaline myopathy

Nemaline myopathies (NMs) are a clinically heterogenous group of non-dystrophic congenital muscle diseases caused by mutations in at least 13 genes. NMs are characterised by muscle weakness, hypotonia and respiratory insufficiency, with presence of cytoskeletal protein aggregates (rods) in skeletal muscle fibers. In the present study we investigated whether neuromuscular junctions (NMJ) are affected in ACTA1-related nemaline myopathy, which accounts for about 50% of severe cases of NMs. For this purpose, we analysed the morphology and function of NMJs in the Acta1(H40Y) knock-in mouse model of the disease.

NEW GENES AND DISEASES: EP.301 The alpha2-subunit of the AP2 clathrin adaptor as the causal gene in an atypical myopathy with granulofilamentous inclusions

Clathrin-mediated endocytosis is the best characterized process for the entry of proteins and lipids at the plasma membrane of eukaryotic cells. We have previously shown that at costameres, muscle adhesion and force transmission sites, clathrin recruited by the AP2 adaptor forms flat plaques required for actin and desmin intermediate filament organization. Here, we describe a patient presenting a progressive muscular atrophy with loss of ambulation, complete tetraplegia, ophthalmoplegia, severe scoliosis and lower limb contractures and no cardiac involvement. Electron microscopy of muscle biopsy revealed inclusions composed of filamentous material, vacuoles, honeycomb structures and thin granular material surcharge. This case was classified as a myofibrillar myopathy with granulofilamentous inclusion bodies despite no mutation in DES, FHL1, CRYAB and other usual suspect genes. Whole exome sequencing identified two compound heterozygote single-nucleotide variants in the AP2A2 gene encoding the AP2 a2-subunit. Immunofluorescent labeling on muscle sections displayed intracellular aggregates positive for AP2, clathrin and desmin. Depletion of total AP2 in mouse muscle induced a dystrophic phenotype with severe force loss, subsarcolemmal disorganization, vacuoles and neuromuscular junction defects while specifically depleting AP2 a2-subunit induced small subsarcolemmal disorganization and light neuromuscular junction defects. Lastly, transferrin uptake assays of endocytosis function in the patient fibroblasts showed a decrease in endocytosis rates compared to fibroblasts from his healthy parents. Altogether, our results suggest that AP2A2 could be the causal gene in this family and AP2A2 mutations leading to a severe but atypical myopathy. Mutations in AP2A2 could represent the first case in a wider family of atypical myopathies with desmin inclusions caused by mutations in genes encoding proteins of the endocytosis machinery.

Optogenetic modeling of human neuromuscular circuits in Duchenne muscular dystrophy with CRISPR and pharmacological corrections.

Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abnormalities contribute to DMD pathogenesis remains unclear. Here, we combine transcriptome analysis and modeling of DMD patient-derived neuromuscular circuits with CRISPR-corrected isogenic controls in compartmentalized microdevices. We show that NMJ volumes and optogenetic motor neuron–stimulated myofiber contraction are compromised in DMD neuromuscular circuits, which can be rescued by pharmacological inhibition of TGFβ signaling, an observation validated in a 96-well human neuromuscular circuit coculture assay. These beneficial effects are associated with normalization of dysregulated gene expression in DMD myogenic transcriptomes affecting NMJ assembly (e.g., MUSK) and axon guidance (e.g., SLIT2 and SLIT3). Our study provides a new human microphysiological model for investigating NMJ defects in DMD and assessing candidate drugs and suggests that enhancing neuromuscular connectivity may be an effective therapeutic strategy.

Persistent neuromuscular junction transmission defects in adults with spinal muscular atrophy treated with nusinersen

ObjectiveSpinal muscular atrophy (SMA) is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. Prior work in models and patients has demonstrated electrophysiological and morphological...

Neuromuscular junction-on-a-chip: ALS disease modeling and read-out development in microfluidic devices.

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease affecting upper and lower motor neurons with no cure available. Clinical and animal studies reveal that the neuromuscular junction (NMJ), a synaptic connection between motor neurons and skeletal muscle fibers, is highly vulnerable in ALS and suggest that NMJ defects may occur at the early stages of the disease. However, mechanistic insight into how NMJ dysfunction relates to the onset and progression of ALS is incomplete, which hampers therapy development. This is, in part, caused by a lack of robust in vitro models. The ability to combine microfluidic and induced pluripotent stem cell (iPSC) technologies has opened up new avenues for studying molecular and cellular ALS phenotypes in vitro. Microfluidic devices offer several advantages over traditional culture approaches when modeling the NMJ, such as the spatial separation of different cell types and increased control over the cellular microenvironment. Moreover, they are compatible with 3D cell culture, which enhances NMJ functionality and maturity. Here, we review how microfluidic technology is currently being employed to develop more reliable in vitro NMJ models. To validate and phenotype such models, various morphological and functional read-outs have been developed. We describe and discuss the relevance of these read-outs and specifically illustrate how these read-outs have enhanced our understanding of NMJ pathology in ALS. Finally, we share our view on potential future directions and challenges.

Whole-mount Staining of Mouse Diaphragm Neuromuscular Junctions.

The neuromuscular junction (NMJ) is a specialized synapse that connects the terminal end of a motor neuron and a skeletal muscle fiber. Defects in NMJ cause abnormalities of neuromuscular transmission, leading to NMJ disorders. The mammalian diaphragm muscle is essential for respiration and has been widely used to study NMJ formation. Here, we provide a simple and straightforward protocol for preparing diaphragms from embryonic, neonatal, and adult mice and for subsequent NMJ staining.

Lipin 1 deficiency causes adult-onset myasthenia with motor neuron dysfunction in humans and neuromuscular junction defects in zebrafish.

Lipin 1 is an intracellular protein acting as a phosphatidic acid phosphohydrolase enzyme controlling lipid metabolism. Human recessive mutations in LPIN1 cause recurrent, early-onset myoglobinuria, a condition normally associated with muscle pain and weakness. Whether and how lipin 1 deficiency in humans leads to peripheral neuropathy is yet unclear. Herein, two novel compound heterozygous mutations in LPIN1 with neurological disorders, but no myoglobinuria were identified in an adult-onset syndromic myasthenia family. The present study sought to explore the pathogenic mechanism of LPIN1 in muscular and neural development.Methods: The clinical diagnosis of the proband was compared to the known 48 cases of LPIN1 recessive homozygous mutations. Whole-exome sequencing was carried out on the syndromic myasthenia family to identify the causative gene. The pathogenesis of lipin 1 deficiency during somitogenesis and neurogenesis was investigated using the zebrafish model. Whole-mount in situ hybridization, immunohistochemistry, birefringence analysis, touch-evoke escape response and locomotion assays were performed to observe in vivo the changes in muscles and neurons. The conservatism of the molecular pathways regulated by lipin 1 was evaluated in human primary glioblastoma and mouse myoblast cells by siRNA knockdown, drug treatment, qRT-PCR and Western blotting analysis.Results: The patient exhibited adult-onset myasthenia accompanied by muscle fiber atrophy and nerve demyelination without myoglobinuria. Two novel heterozygous mutations, c.2047A>C (p.I683L) and c.2201G>A (p.R734Q) in LPIN1, were identified in the family and predicted to alter the tertiary structure of LPIN1 protein. Lipin 1 deficiency in zebrafish embryos generated by lpin1 morpholino knockdown or human LPIN1 mutant mRNA injections reproduced the myotomes defects, a reduction both in primary motor neurons and secondary motor neurons projections, morphological changes of post-synaptic clusters of acetylcholine receptors, and myelination defects, which led to reduced touch-evoked response and abnormalities of swimming behaviors. Loss of lipin 1 function in zebrafish and mammalian cells also exhibited altered expression levels of muscle and neuron markers, as well as abnormally enhanced Notch signaling, which was partially rescued by the specific Notch pathway inhibitor DAPT.Conclusions: These findings pointed out that the compound heterozygous mutations in human LPIN1 caused adult-onset syndromic myasthenia with peripheral neuropathy. Moreover, zebrafish could be used to model the neuromuscular phenotypes due to the lipin 1 deficiency, where a novel pathological role of over-activated Notch signaling was discovered and further confirmed in mammalian cell lines.