Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

Abstract

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

Fig. 1

Mutations in AGRN Gene Causing CMS. a Family 1 and 2 pedigrees. b, c EMG reveals obvious decremental response of the compound muscle-fiber action potential (CMAPs) in Patient 1 and Patient 2.1. d Whole-mount preparations of muscle biopsy stained with α-bungarotoxin in red and stained for axon terminals with neurofilament in green. In control, the axonal branch classically ends as a fork and innervates a well-defined synaptic structure. In Patient 1, the neurofilament staining showed frequent presynaptic sprouting (arrowhead). Scale 10 µm. e Domain organization of agrin and recombinant mini-agrin used in this study and position of the L1, L2 and LM mutations. Agrin is a mosaic protein composed of the following domains: SS, signal peptide; NtA, N-terminal laminin-binding domain, red; TM, trans-membrane domain; FS, follistatin-like, blue; LE, laminin EGF-like, orange; S/T, serine/threonine-rich region, yellow; SEA, Sea urchin sperm protein, Enterokinase and Agrin motif, purple; EG, EGF-like, red; LG, laminin-globular-like, green; LG2 domain interacts with α-Dystroglycan and integrins; Sites of alternative splicing: SS-NtA or TM at the 5’end, A/y in the LG2 domain, B/z in the LG3 domain. f Multiple alignments of amino acid sequences in the agrin LG2 domains (residues 1949–1743 from H. sapiens_NP_940978) show the positions of L1, L2 and LM mutations

Fig. 2

Abnormal subcellular localizations of mutant mini-agrin in primary motoneurons. a Representative confocal images of MNs 2 days after transfection with WT, L1, L2 or LM mini-agrin IRES eGFP (green) constructs. Overexpressed human agrins were revealed with an anti-human-agrin antibody (red). b Magnification of the cell bodies from images in a. WT agrin showed a diffuse distribution often with stronger labeling at the soma/neurite boundary characteristic of secreted proteins (arrow). L2 agrin accumulates exclusively in the soma (star). c Magnification of the axonal projections from images in a. L1 and LM agrin accumulate along the axo-dendritic compartment (white arrowhead). Scale bars: 20 μm

Fig. 3

Mutant mini-agrin are mislocalized in motoneurons in vivo. a Confocal images of spinal cord cryosection from chick neural tube electroporated with WT, L1, L2 or LM mini-agrin IRES eGFP constructs. Electroporated neurons are identified by eGFP (green), and human agrin immunostaining (red). Nuclei are stained with DAPI (blue). Scale bar: 200 µm. White boxes show the enlarged region. b Higher magnification on the anterior horn shows the accumulation of the three mutant agrins in the soma of MNs. Scale bar: 50 µm. c, d Confocal images of axonal projections from electroporated motoneurons in the upper limb revealed by eGFP show focal accumulations of L1 et LM but not L2 mutants along the axon projections. Scale bar: 20 µm. White boxes show regions enlarged in d

Fig. 4

Mutant mini-agrins accumulate in the endoplasmic reticulum and are less secreted. a Confocal images of SHEP cells transfected with agrin IRES eGFP (green) counterstained for agrin (red) and Golgi apparatus (GM130, white). Full white arrowheads show agrin enrichment at the Golgi apparatus. Empty white arrowheads indicate the absence of agrin in the Golgi apparatus. Scale bar: 10 μm. b Confocal images of SHEP cells transfected with agrin IRES eGFP counterstained for agrin and an endoplasmic reticulum marker KDEL (white). Scale bar: 10 μm. c, d Quantification of agrin/Golgi co-localization and agrin/endoplasmic reticulum (ER) co-localization (Kruskal–Wallis test followed by Dunn's multiple comparison test; **p < 0.01; ***p < 0.001). e Representative dot-blot for agrin quantification in conditioned medium (CM) and whole-cell extract (WCE) from cultures expressing WT, L1, L2 or LM mini-agrins. f Graphic representation of the ratio of secreted agrin quantified from the dot-blots and at least three independent experiments (one-way ANOVA followed by Dunnett’s test; ***p < 0.001)

Fig. 5

Mutant FL-agrins accumulate in the endoplasmic reticulum and are less secreted. a. Confocal images of SHEP transfected cells with FL-agrin fused to eGFP (in green), counterstained with the endoplasmic reticulum marker KDEL (in red) and the Golgi apparatus marker GM130 (in white). White arrow shows agrin exclusion from the Golgi apparatus. Scale bar: 10 μm. b Quantification of agrin/Golgi co-localization (Kruskal–Wallis test followed by Dunn's multiple comparison test; **p < 0.01). c Quantification of agrin/endoplasmic reticulum (ER) co-localization (Kruskal–Wallis test followed by Dunn's multiple comparison test; *p < 0.05). d Representative dot-blot for agrin quantification in conditioned medium (CM) and whole-cell extract (WCE) from cultures expressing WT, L1, L2 or LM FL-agrins. e Graphic representation of FL-agrin secreted ratio quantified from the dot-blots and at least three independent experiments (one-way ANOVA followed by Dunnett's multiple comparison test; ***p < 0.001). c Quantification of the percentage of FL agrin that colocalized with the Golgi apparatus marker GM130 (Kruskal–Wallis test followed by Dunn's multiple comparison test; **p < 0.01)

Fig. 6

Mutant agrins trigger unfolded protein response. a, b Quantification of the size and intensity of KDEL immunostaining on SHEP cell confocal images after expression of mini or FL-agrins (n = 3; Kruskal–Wallis test followed by Dunn's multiple comparison test). c Co-immunoprecipitation of mini and FL-agrins with GRP78/BIP. IP agrin: agrin immunoprecipitation. α indicates the antibody used for the western blot.). d Gel electrophoresis resolving the unspliced (uXBP1) and spliced forms (sXBP1) of XBP1 and densitometric analysis of u/sXBP1 band intensities relative to the total (uXBP1 + sXBP1) XBP1, (n = 3; one-way ANOVA followed by Dunnett's Multiple Comparison Test). e–i GRP78, GRP94, CHOP, GADD34, and EDEM1 mRNA quantification by RT-qPCR on HEK293T transfected with WT or mutant mini-agrin expression vectors or empty vector in control (n = 3; one-way ANOVA followed by Dunnett's multiple comparison test to compare all mutant agrins to WT agrin; *p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 7

Mutant agrins trigger apoptosis in vitro and in ovo. a Representative images of SHEP cultures co-expressing mini-agrin and eGFP (green). Blue: nuclei are stained with DAPI. Red: activated caspase-3 immunostaining. Scale bar: 200 µm. b–d Quantification of activated caspase-3 positive cells and mean cell area 48 h after transfection, and quantification of survival at 72 h (n = 3; One-way ANOVA followed by Dunnett's Multiple Comparison Test). e Representative confocal images of activated caspase-3 immunostaining (red) in electroporated neurons co-expressing mini-agrin and eGFP (green) in ovo. Scale bar: 100 µm. f Quantification of activated caspase-3 positive neurons in spinal cord cryosections spanning the entire electroporated spinal cord area (n = 6 embryos per condition from 3 independent electroporation sessions; one-way ANOVA followed by Dunnett's multiple comparison test). CL corresponds to the same quantification on the contralateral side of the spinal cord that did not receive the transgene

Fig. 8

Conditioned media from cells expressing full-length mutant agrins induce less AChR clustering in myotubes. a–c Pictures of C2C12 myotubes incubated with conditioned media from cells transfected with WT or mutant agrins. Negative control (CTL −) corresponds to a conditioned culture media from cells transfected with an empty vector. AChR clusters were stained with Alexa Fluor 488-conjugated to α-BTX. Scale bar: 50 μm. Myotubes exposed to the same volume of condition media (a), or the same amount of secreted agrin (b), or the same amount of intracellular agrin from the whole-cell extract resuspended in fresh medium (c). Scale bar: 20 µm. d–f Quantification of the number of AChR clusters per field normalized to the WT agrin condition for the same volume of condition media (d), or the same amount of secreted agrin (e), or the same amount of intracellular agrin (f). Positive control (CTL +) corresponds to C2C12 myotubes treated with 2 nM of purified WT human agrin. Three independent experiments. One-way ANOVA followed by Dunnett's multiple comparison test (***p < 0.001)

Fig. 9

Patient 1 hiPSC-derived motoneurons accumulate mutant agrin, induce less AChR clustering, and show reduced survival rate in co-culture. a Representative images of hiPSC-derived MNs after 25 days of differentiation immunostained for Islet1 (red), agrin (green), and Tuj1 (white). Scale bar: 20 µm. b, c Percentage of surviving hiPSC-derived MNs after 14 and 25 days of differentiation and automatically quantified after immunostaining for Islet1. Data are presented as mean ± SEM, three independent experiments, in quadruplicate and were analyzed by a Student t test. d, e Quantification of the somatic and neuritic intensity and area of agrin staining in hiPSC-derived MNs after 25 days of differentiation. The data were normalized on the number of hiPSC-derived MNs and presented as mean ± SEM, three independent experiments in quadruplicate were analyzed with a Student’s t test. f, g Quantification by RT-qPCR of unspliced (uXBP1) or spliced XBP1 (sXBP1) mRNA level normalized by the total XBP1 (tXBP1) and then by the control MNs at D25. Data are presented as mean ± SD, three independent experiments in triplicate were analyzed by a t test (ns p > 0.05; ***p < 0.001) h Representative images of co-cultures between hiPSC-derived MNs and human primary skeletal muscle cells for 4 days. Co-cultures were characterized by immunostaining for Tuj1, myosin heavy chain (MF20), and AChR. Scale bar: 50 µm. i, j Quantification of the number and the area of AChR clusters determined by immunostaining with AChR antibody. The number and the area of AChR clusters were normalized on myotube area determined by immunostaining for myosin heavy chain. Data presented as mean ± SEM, three independent experiments in quadruplicate and were analyzed by Student t Test. k Based on the data generated in panel h, and cluster size distribution was determined in function of the area. Data are presented as mean ± SEM, three independent experiments in quadruplicate and were analyzed by Student’s t test. l Number of hiPSC-derived MNs after 14 days of differentiation and automatically quantified after immunostaining for Islet1. Data are presented as mean ± SEM, three independent experiments, in quadruplicate, and were analyzed by a two-way ANOVA test