Congenital myasthenic syndrome–associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner

Ohkawara, Bisei; Shen, Xin Ming; Selcen, Duygu; Nazim, Mohammad; Bril, Vera; Tarnopolsky, Mark A.; Brady, Lauren; Fukami, Sae; Amato, Anthony A.; Yış, Uluç; Ohno, Kinji; Engel, Andrew G.

doi:10.1172/jci.insight.132023

Cited by 15 publications

(30 citation statements)

References 39 publications

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“…Given the modulable nature of NT enzymatic activity, its effect on Agrin signalling, and its involvement in processes of synaptic reorganisation, it is surprising that deregulation of the NT-agrin axis has been linked only to a small number of pathological states 11,40,41,48 . One of the few known pathogenic implications of NT deregulation is centred on the formation and maintenance of NMJ’s.…”

Section: Discussionmentioning

confidence: 99%

NT-mini, a recombinant tool for the study of Neurotrypsin functionality

Canciani

Capitanio

Stanga

et al. 2020

Preprint

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Neurotrypsin (NT) is a highly specific nervous system multi-domain serine-protease best known for its selective processing of the potent synaptic organiser agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. Something which, based on the available literature, likely stems from NT-based regulation of agrin signalling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, owing to the scarce molecular detail regarding NT activity and NT-agrin interactions. The difficult recombinant production of NT in its catalytically competent form is at the base of these limitations, and is currently constraining a more detailed molecular, biochemical and structural characterisation of the NT-agrin system. We have developed a novel strategy to reliably produce and purify a truncated but catalytically competent variant of NT called NT-mini. The characterisation of our construct highlighted almost wild type-like behaviour with comparable specificity, and conservation of modulation by calcium and heparin despite the lack of several accessory domains. With the data obtained from NT-mini it was then possible to identify NT's heparin-binding domain, and discover a novel putative Zinc-based modulation of NT. Additionally, NT-mini allowed us to investigate the effect of NT activity on myotube formation in controlled cell-based experiments, evidencing a negative impact on myoblast fusion dependant on enzymatic activity. Collectively, this shows the viability of NT-mini as a model to study NT functionality, allowing to expand both in-vitro and "in-cellulo" investigations and providing a foundation to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

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Section: Discussionmentioning

confidence: 99%

NT-mini, a recombinant tool for the study of Neurotrypsin functionality

Canciani

Capitanio

Stanga

et al. 2020

Preprint

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“…The sperm protein, enterokinase, and agrin (SEA) domain is important for the glycosylation and secretion of agrin, and also protects agrin from degradation by proteases such as neurotrypsin [ 41 ]. Indeed, p.L1176P [ 41 ] and p.S1180L [ 44 ] in the SEA domain cause the instability of the agrin protein and impair the formation of AChR clusters in C2C12 myotubes. Between the NtA and SEA domains, nine follistatin (FS), two laminin EGF-like (LE), and one serine/threonine-rich (S/T) domains exist.…”

Section: Agrin-lrp4-musk Signaling Pathwaymentioning

confidence: 99%

“…The first and second laminin G-like domains (LG1 and LG2) are essential for anchoring agrin to the NMJ using α-dystroglycan or other muscle-specific cell surface molecule(s) [ 44 , 47 ]. Two mutations (p.P1448L and p.G1509W) in the LG1 domain lead to CMS [ 43 , 44 ].…”

Section: Agrin-lrp4-musk Signaling Pathwaymentioning

confidence: 99%

Secreted Signaling Molecules at the Neuromuscular Junction in Physiology and Pathology

Ohkawara

Ito

Ohno

2021

IJMS

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: Signal transduction at the neuromuscular junction (NMJ) is affected in many human diseases, including congenital myasthenic syndromes (CMS), myasthenia gravis, Lambert–Eaton myasthenic syndrome, Isaacs’ syndrome, Schwartz–Jampel syndrome, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. The NMJ is a prototypic cholinergic synapse between the motor neuron and the skeletal muscle. Synaptogenesis of the NMJ has been extensively studied, which has also been extrapolated to further understand synapse formation in the central nervous system. Studies of genetically engineered mice have disclosed crucial roles of secreted molecules in the development and maintenance of the NMJ. In this review, we focus on the secreted signaling molecules which regulate the clustering of acetylcholine receptors (AChRs) at the NMJ. We first discuss the signaling pathway comprised of neural agrin and its receptors, low-density lipoprotein receptor-related protein 4 (Lrp4) and muscle-specific receptor tyrosine kinase (MuSK). This pathway drives the clustering of acetylcholine receptors (AChRs) to ensure efficient signal transduction at the NMJ. We also discuss three secreted molecules (Rspo2, Fgf18, and connective tissue growth factor (Ctgf)) that we recently identified in the Wnt/β-catenin and fibroblast growth factors (FGF) signaling pathways. The three secreted molecules facilitate the clustering of AChRs by enhancing the agrin-Lrp4-MuSK signaling pathway.

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“…CMS lead to disabling fatigable muscle weakness and can be fatal due to respiratory muscle weakness. Mutations in over 30 genes are known to be causative for CMS, and at least 16 have been identified in the AGRN gene (Finsterer, 2019;Ohkawara et al, 2020). AGRN mutations can impact AChR clustering by decreasing the phosphorylation of MuSK, degrading secreted neural AGRIN, and impairing the anchoring of AGRIN to the sarcolemma (Ohkawara et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…Mutations in over 30 genes are known to be causative for CMS, and at least 16 have been identified in the AGRN gene (Finsterer, 2019;Ohkawara et al, 2020). AGRN mutations can impact AChR clustering by decreasing the phosphorylation of MuSK, degrading secreted neural AGRIN, and impairing the anchoring of AGRIN to the sarcolemma (Ohkawara et al, 2020). Patients with AGRN mutations present with exercise-induced proximal or distal muscle weakness, and varying degrees of ptosis, ophthalmoplegia, facial weakness, and respiratory muscle weakness.…”

Section: Introductionmentioning

confidence: 99%

Modulation of the Acetylcholine Receptor Clustering Pathway Improves Neuromuscular Junction Structure and Muscle Strength in a Mouse Model of Congenital Myasthenic Syndrome

Spendiff

Howarth

McMacken

et al. 2020

Front. Mol. Neurosci.

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Introduction: Congenital myasthenic syndromes (CMS) are a diverse group of inherited neuromuscular disorders characterized by a failure of synaptic transmission at the neuromuscular junction (NMJ). CMS often present early with fatigable weakness and can be fatal through respiratory complications. The AGRN gene is one of over 30 genes known to harbor mutations causative for CMS. In this study, we aimed to determine if a compound (NT1654), developed to stimulate the acetylcholine receptor (AChR) clustering pathway, would benefit a mouse model of CMS caused by a loss-of-function mutation in Agrn (Agrnnmf380 mouse).Methods:Agrnnmf380 mice received an injection of either NT1654 or vehicle compound daily, with wild-type litter mates used for comparison. Animals were weighed daily and underwent grip strength assessments. After 30 days of treatment animals were sacrificed, and muscles collected. Investigations into NMJ and muscle morphology were performed on collected tissue.Results: While minimal improvements in NMJ ultrastructure were observed with electron microscopy, gross NMJ structure analysis using fluorescent labelling and confocal microscopy revealed extensive postsynaptic improvements in Agrnnmf380 mice with NT1654 administration, with variables frequently returning to wild type levels. An improvement in muscle weight and myofiber characteristics helped increase forelimb grip strength and body weight.Conclusions: We conclude that NT1654 restores NMJ postsynaptic structure and improves muscle strength through normalization of muscle fiber composition and the prevention of atrophy. We hypothesize this occurs through the AChR clustering pathway in Agrnnmf380 mice. Future studies should investigate if this may represent a viable treatment option for patients with CMS, especially those with mutations in proteins of the AChR clustering pathway.

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Congenital myasthenic syndrome–associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner

Cited by 15 publications

References 39 publications

NT-mini, a recombinant tool for the study of Neurotrypsin functionality

NT-mini, a recombinant tool for the study of Neurotrypsin functionality

Secreted Signaling Molecules at the Neuromuscular Junction in Physiology and Pathology

Modulation of the Acetylcholine Receptor Clustering Pathway Improves Neuromuscular Junction Structure and Muscle Strength in a Mouse Model of Congenital Myasthenic Syndrome

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