1. Neuroscience
Download icon

A mechanism in agrin signaling revealed by a prevalent Rapsyn mutation in congenital myasthenic syndrome

  1. Guanglin Xing
  2. Hongyang Jing
  3. Lei Zhang
  4. Yu Cao
  5. Lei Li
  6. Kai Zhao
  7. Zhaoqi Dong
  8. Wenbing Chen
  9. Hongsheng Wang
  10. Rangjuan Cao
  11. Wen-Cheng Xiong
  12. Lin Mei  Is a corresponding author
  1. Case Western Reserve University, United States
  2. Augusta University, United States
  3. Louis Stokes Cleveland Veterans Affairs Medical Center, United States
Research Article
  • Cited 5
  • Views 1,025
  • Annotations
Cite this article as: eLife 2019;8:e49180 doi: 10.7554/eLife.49180

Abstract

Neuromuscular junction is a synapse between motoneurons and skeletal muscles, where acetylcholine receptors (AChRs) are concentrated to control muscle contraction. Studies of this synapse have contributed to our understanding of synapse assembly and pathological mechanisms of neuromuscular disorders. Nevertheless, underlying mechanisms of NMJ formation was not well understood. To this end, we took a novel approach – studying mutant genes implicated in congenital myasthenic syndrome (CMS). We showed that knock-in mice carrying N88K, a prevalent CMS mutation of Rapsyn (Rapsn), died soon after birth with profound NMJ deficits. Rapsn is an adapter protein that bridges AChRs to the cytoskeleton and possesses E3 ligase activity. In investigating how N88K impairs the NMJ, we uncovered a novel signaling pathway by which Agrin-LRP4-MuSK induces tyrosine phosphorylation of Rapsn, which is required for its self-association and E3 ligase activity. Our results also provide insight into pathological mechanisms of CMS.

https://doi.org/10.7554/eLife.49180.001

Introduction

The neuromuscular junction (NMJ) is a synapse between motoneurons and muscle fibers. At vertebrate NMJs, motor nerve terminals release acetylcholine (ACh), which activates ACh receptors (AChRs) concentrated on postsynaptic membranes to initiate muscle contraction. The concentration of AChR at postsynaptic membrane requires Agrin, a factor released from motoneurons (McMahan, 1990), which binds to LRP4 (Kim et al., 2008; Zhang et al., 2008) to activate MuSK (DeChiara et al., 1996; Glass et al., 1996; Jennings et al., 1993). Downstream effector of MuSK is believed to be Rapsn, a cytoplasmic protein that is required for AChR clustering and NMJ formation. Rapsn, discovered as a peripheral membrane protein associated with AChRs in the electric organ of Torpedo (Cohen et al., 1972; Neubig and Cohen, 1979; Porter and Froehner, 1985), colocalizes with AChRs at developing as well as adult NMJs (Froehner et al., 1981; Noakes et al., 1993; Sealock et al., 1984). It could induce AChR clusters in heterologous cells (Froehner et al., 1990; Li et al., 2016; Phillips et al., 1991); Rapsn null mutant mice die soon after birth without AChR clusters (Gautam et al., 1995), indicating a critical role in NMJ formation. Being a classic adapter protein, Rapsn is thought to bridge the AChR to the cytoskeleton (Apel et al., 1995; Bartoli et al., 2001; Chen et al., 2016; Lee et al., 2009; Maimone and Merlie, 1993; Miyazawa et al., 1999; Ramarao et al., 2001; Ramarao and Cohen, 1998). We showed recently that Rapsn possesses E3 ligase activity (Li et al., 2016). Mutation of a cysteine residue necessary for the E3 ligase activity impairs its ability to cluster AChRs in vitro and to form NMJs in knock-in mice, suggesting that Rapsn may also be a signaling molecule. Nevertheless, how signal is transduced from upstream molecules such as Agrin-LRP4-MuSK to Rapsn, remains unclear, a glaring gap in our understanding of NMJ formation.

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of NMJ diseases caused by mutations of genes of NMJ structure and function proteins (Engel et al., 2003; Engel et al., 2015; Engel and Sine, 2005; Hantaï et al., 2004). Based on the primary deficits, CMSs could be classified into presynaptic, postsynaptic, and synaptic cleft-associated groups (Engel et al., 2003; Engel et al., 2015; Engel and Sine, 2005). A majority of CMS cases, ~75%, involve mutations of genes encoding proteins for postsynaptic development or function; and among them, mutations in AChR subunits and Rapsn are prevalent (Engel et al., 2003; Engel et al., 2015; Engel and Sine, 2005). Although more than thirty genes have been identified in CMS patients, mouse models to mimic such mutations are rare. Hence underlying pathological mechanisms remain not well understood. Presently, about 40 mutations in the Rapsn gene have been identified in patients with CMSs, accounting for 15% of total CMS cases (Dunne and Maselli, 2003; Maselli et al., 2007; Maselli et al., 2003; Milone et al., 2009; Müller et al., 2004; Ohno and Engel, 2004; Ohno et al., 2002; Ohno et al., 2003; Yasaki et al., 2004). Of them, the N88K (asparagine 88 to lysine) mutation is the most common (Milone et al., 2009; Müller et al., 2003). About 90% of Rapsn-related CMS patients carry N88K mutation (Burke et al., 2003; Milone et al., 2009; Müller et al., 2003). Patients are either homozygous for N88K or heteroallelic for N88K with another Rapsn mutation. Pathological examination of patient specimens revealed attenuated or fragmented AChR clusters and reduced Rapsn protein, and altered junctional folds (Maselli et al., 2003; Milone et al., 2009; Ohno et al., 2002). In severe cases, N88K heteroallelic with c.966 + 1GT, L14P, or a frameshift mutation may cause postnatal death (Maselli et al., 2003; Milone et al., 2009; Richard et al., 2003). N88K mutant Rapsn induced fewer AChR clusters, compared with wild type Rapsn in heterologous cells (Ohno et al., 2002) and was unable to fully rescue Agrin-induced AChR clusters in Rapsn mutant cultured myotubes (Cossins et al., 2006).

To understand molecular mechanisms of NMJ formation, we studied CMS mutations of the Rapsn gene with an idea that such mutations with clinic implications would reveal unexpected insight. In particular, we generated N88K knock-in mice to investigate the in vivo impact of N88K mutation on the NMJ. N88K mutant mice displayed profound deficits not only in AChR clusters, but also in nerve terminals at both light and electron microscopic levels. Developmentally, the NMJ abnormality was observed as early as embryonic day 14. We performed a combination of cell and molecular biological experiments including studying N88K mutant muscles. We demonstrated that N88K mutation inhibited the E3 ligase activity of Rapsn by reducing its phosphorylation and self-association. Our results not only unravel pathophysiological mechanisms of N88K mutation, but also provide novel insight into how signals are transduced from Agrin-LRP4-MuSK to Rapsn.

Results

Aberrant NMJ formation in N88K mt mice

To study pathological mechanisms of the CMS-associated mutation N88K, we generated N88K knock-in mutant (mt) mice by CRISPR-Cas9 technique (Figure 1—figure supplement 1A and B). The mRNA of N88K Rapsn was comparable to that of wild type (WT) Rapsn (Figure 1—figure supplement 1C). The protein levels of Rapsn were similar between muscle lysates of the two genotypes (Figure 1A); and the mutation seemed to have little effect on the levels of ubiquitinated Rapsn (Figure 1—figure supplement 1D and E). However, N88K homozygous mt mice died within 24 hr of birth with cyanosis, suggesting respiratory failure. To examine the effect of N88K mutation on the NMJ, diaphragms were isolated from neonatal P0 mice and stained for AChR and nerve terminals. In WT diaphragms, AChR clusters were abundant and localized in the middle of muscle fibers, whereas in Rapsn null mt (-/-) mice, there was almost no AChR cluster (Figure 1B–D). N88K mt mice were able to form AChR clusters, but their number and size were dramatically reduced (Figure 1C–F and Figure 1—figure supplement 1F). Remaining clusters were elongated, in contrast to oval plaque morphology in WT mice (Figure 1D and G and Figure 1—figure supplement 1G) and distributed in a wider region of muscle fibers (Figure 1C and H). AChR clusters in N88K mt mice were reduced in staining intensity (Figure 1I), indicating reduced AChR concentration. In addition, N88K mt mice displayed abnormal nerve terminal arborization, with increased numbers of secondary, tertiary, quaternary branches, as observed in null mt mice (Figure 1B–D and J and Figure 1—figure supplement 1H). To eliminate potential off-target effects of CRISPR-Cas9, we generated N88K/- mice that possess null mutation on one chromosome and N88K mutation on the other. They exhibited similar NMJ deficits to N88K mt mice (Figure 1B–J and Figure 1—figure supplement 1F–H), suggesting that NMJ deficits in N88K mt mice are likely due to N88K mutation, not an off-target effect.

Figure 1 with 1 supplement see all
Few AChR clusters and extensive nerve terminal arborization in N88K mt mice.

(A) Comparable Rapsn protein level between WT and N88K mt mice. Tissue lysates from WT, N88K mt, -/- (Rapsn null mt) diaphragms were subjected to western blotting with anti-Rapsn, and anti-α-AChR antibodies, using GAPDH as loading control. Note that α-AChR protein level was not altered in N88K and -/-, compared with WT controls. (B) Reduced AChR clusters and extensive axonal arborization in N88K mt mice. P0 diaphragms from WT, N88K, N88K/-, -/- mice were stained whole-mount with Flour 594-α-BTX (red) to label AChR clusters and with anti-NF/Syn antibodies (green) to label motor nerve terminals. V, ventral. L, left. (C, D) Higher magnification views of AChR clusters and motor axon branches. The boundary of AChR clusters was indicated by dashed blue lines. Heatmap, AChR intensity; arrows, elongated AChR clusters along axons. (E–J) Quantitative analysis of AChR cluster number (E), AChR cluster size (F), AChR cluster length (G), endplate band width (H), AChR fluorescence intensity (I), secondary branch length (J). Data were shown as mean ± SEM; **, p<0.01; ***, p<0.001, One-way ANOVA, n = 6 mice. Also see Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.49180.002

At electron microscopic (EM) level, in WT mice, axon terminals were filled with abundant synaptic vesicles, some of which were clustered at active zones (Figure 2A and C). Synaptic clefts were filled with synaptic basal lamina; and, on the postsynaptic side, junctional folds were observable (Figure 2A and C). However, junctional folds in N88K mt mice were fewer and the remainders were shorter than those of controls (Figure 2B,D and E). N88K mt mice also showed reduced number of synaptic vesicles in axon terminals (Figure 2F), compared with WT controls. The vesicle diameters and synaptic cleft width were similar between two genotypes (Figure 2G and H). Taken together, studies of P0 mice revealed both pre- and post- deficits at the NMJ, providing a mechanism of neonatal lethality of N88K mutation.

Small junctional folds, reduced vesicle density and diminished aneural AChR clusters in N88K mt mice.

(A, B) Low magnification NMJ EM images of WT and N88K mt, including synaptic vesicles (SVs), synaptic cleft, synaptic basal lamina (SBL), and postsynaptic junctional folds (JF). M, muscle; N, nerve; (C, D) High magnification images of SVs, SBL, and JF. Note that junctional folds were smaller in N88K mt, and vesicle density was reduced in N88K mt, compared with WT controls. Asterisks indicate active zone. (E–H) Quantitative data of junctional fold length (E), synaptic vesicle density (F), vesicle diameter (G), synaptic cleft width (H) of WT and N88K mt. Data were shown as mean ± SEM; ***, p<0.001, unpaired t-test, n = 3 mice. (I–L) Few aneural AChR clusters in N88K mt mice at E14. E14 diaphragms from WT, N88K mt mice were stained whole-mount with Flour 594-α-BTX (red) to label AChR clusters and with anti-NF/Syn (green) antibodies to label motor nerve terminals. (I, J) Low magnification view. (K, L) High magnification view. (M, N) Quantitative data of endplate band width and AChR cluster number. Data were shown as mean ± SEM, ***, p<0.001, unpaired t-test, n = 3 mice.

https://doi.org/10.7554/eLife.49180.006

Muscles form primitive, aneural AChR clusters in advance of the arrival of motor nerve terminals (Lin et al., 2001; Yang et al., 2001b; Luo, 2010). Innervation induces the formation of new AChR clusters, perhaps by enlarging some of the aneural clusters. The formation of aneural clusters requires LRP4, MuSK, and Rapsn, but not Agrin (Lin et al., 2001; Remédio et al., 2016; Yang et al., 2001b). Having observed NMJ deficits in neonatal mice, we wondered whether the N88K mutation alters aneural AChR clusters and dissected diaphragms from embryos at E14, when axon terminals start branching out to innervate muscle fibers (Li et al., 2018; Lin et al., 2001; Wu et al., 2010; Yang et al., 2001b). As shown in Figure 2I and K, aneural AChR clusters of WT mice were numerous and elongated in shape (Figure 2M and N). Most of them were not innervated although nerve terminals were present. Remarkably, aneural AChR clusters were rarely detectable in N88K mt diaphragms (Figure 2J,L,M and N). These results suggest that N88 is necessary for the formation of aneural clusters and that the AChR clusters at P0 N88K mt diaphragms are likely induced by nerve terminals.

To examine whether synaptic transmission is altered in N88K mt mice, we first recorded resting membrane potentials and found that they were comparable between WT and N88K mt mice (Figure 3A). Next, we measured miniature endplate potentials (mEPP), postsynaptic potentials elicited by spontaneous vesicle release. In WT controls, mEPPs were easily detectable in ~88% of muscle fibers; however, this number was reduced to ~13% in N88K homozygous mt mice (Figure 3B). In addition, mEPP amplitudes and frequencies were reduced in N88K mt mice, compared with WT controls (Figure 3C–E). While N88K heterozygous (i.e., N88K/+) mice showed no detectable mEPP deficits (Figure 3), those in N88K/- and N88K homozygous mice were comparable (Figure 3). These results are in agreement with morphological deficits and indicate neuromuscular transmission is impaired by N88K mutation.

Reduced synaptic transmission in N88K mt mice.

(A) Comparable resting membrane potentials among WT, N88K/+, N88K mt, and N88K/- mt. P0 hemi-diaphragms at ventral, left were recorded. (B) Percentage of muscle fibers with mEPPs in 3 min of recording (n = 120, 6 mice). (C) Representative mEPP traces. (D, E) Reduced mEPP amplitude (D) and reduced mEPP frequency in N88K mt and N88K/- mt (E). Data were shown as mean ± SEM, **, p<0.01, ***, p<0.001, One-way ANOVA, n = 6 mice.

https://doi.org/10.7554/eLife.49180.008

Impaired ability of N88K Rapsn in AChR clustering

Rapsn is able to induce AChR clusters in heterologous cells (Froehner et al., 1981; Li et al., 2016; Phillips et al., 1991). To investigate mechanisms of how N88K mutation reduced AChR concentration at the NMJ, HEK293T cells were transfected with EGFP-tagged WT or N88K Rapsn together with four different AChR subunits (α, β, γ, δ), fixed and stained for AChR clusters. As shown in Figure 4A, AChRs were diffused when coexpressed with EGFP empty vector, but became clustered in cells co-expressing WT Rapsn, and these AChR clusters colocalized with Rapsn clusters, in agreement with previous reports (Froehner et al., 1990; Li et al., 2016; Phillips et al., 1991). N88K was able to form aggregates in HEK293 cells (Figure 4A), in agreement with a previous report (Ohno et al., 2002). However, fewer AChR clusters were observed in HEK293T cells expressing N88K, compared with those expressing WT Rapsn (Figure 4A and B). This result could suggest that the N88K mutation prevents Rapsn from clustering AChRs in HEK293T cells or maintaining AChR surface expression. To determine whether N88K mutation alters surface AChR levels, HEK293T cells were incubated with Sulfo-NHS-SS-biotin at 4°C. Biotin-labeled surface proteins were precipitated and probed for different AChR subunits and transferrin (Trf) as control. As shown in Figure 4C, surface AChR, total AChR and Rapsn levels were comparable between cells transfected with WT and N88K Rapsn, suggesting that N88K mutation did not alter AChR levels on cell surface. Moreover, N88K mutation did not change Rapsn’s stability in HEK293T cells (Figure 4—figure supplement 1A and B). These results suggest that the ability of N88K mt Rapsn to induce AChR clusters was reduced.

Figure 4 with 2 supplements see all
Impaired ability of N88K mt Rapsn in AChR clustering in HEK293T cells and in cultured muscle cells.

(A) Impaired ability of N88K mt Rapsn to induce AChR clustering in HEK293T cells. HEK293T cells were transfected with AChR subunits (α, β, γ, δ), along with EGFP empty vector, Rapsn-EGFP, or N88K-EGFP. After 36 hr, live, unfixed cells were incubated with Flour 594-α-BTX (red) to label surface AChRs. Arrows, AChR clusters colocalized with Rapsn clusters. (B) Quantitative data of (A) (mean ± SEM). ***, p<0.001, unpaired t-test, n = 20 cells. (C) Comparable amount of AChRs and Rapsn in (A). Total levels of α-AChR, β-AChR, Rapsn, and surface α-AChR, β-AChR in the parallel experiment of (A) were examined by western blotting, using GAPDH and Transferrin (Trf) as lysate and surface protein loading controls, respectively. (D) Comparable total Rapsn and AChR, and surface AChR expression in N88K mt cultured myotubes. Total α-AChR, β-AChR, Rapsn and surface α-AChR, β-AChR from WT or N88K mt C2C12 myotubes were examined by western blotting, using GAPDH and Trf as lysate and surface protein loading controls, respectively. (E) Fewer Agrin-induced AChR clusters in N88K mt C2C12 myotubes, compared with WT controls. Myotubes were treated with or without 50 ng/ml Agrin for 12 hr. Arrows, AChR clusters. (F) Quantitative data of (E) (mean ± SEM), ***, p<0.001, Two-way ANOVA, n = 20 cells. Also see Figure 4—figure supplement 1 and Figure 4—figure supplement 2.

https://doi.org/10.7554/eLife.49180.010

Next, to test the ability of N88K mt Rapsn in AChR clustering in muscle cells, we generated N88K mt C2C12 cells using the same CRISPR-Cas9 strategy to generate N88K mt mice. The mutation was confirmed by genomic DNA sequencing (Figure 4—figure supplement 2A). The levels of N88K mRNA (Figure 4—figure supplement 2B) and protein (Figure 4D) in mt C2C12 myotubes were comparable to those of WT Rapsn in control myotubes. The mutation had no apparent effect on the stability of Rapsn protein (Figure 4—figure supplement 2C and D) and surface AChR levels (Figure 4D). However, AChR clusters were reduced in Agrin-treated N88K mt C2C12 myotubes, in contrast to robust AChR clusters in Agrin-treated WT C2C12 myotubes (Figure 4E and F). These results demonstrated that N88K mutation impairs the ability of Rapsn to induce AChR clusters.

Reduced E3 ligase activity of N88K mt Rapsn

As a scaffold protein, Rapsn could induce AChR clusters by bridging AChR subunits to the actin cytoskeleton (Burden et al., 1983; LaRochelle and Froehner, 1986Zhang et al., 2007; Walker et al., 1984). Therefore, we first determined whether the N88K mutation alters Rapsn binding to surface AChRs and cytoplasmic actin. Surface AChRs were purified with biotin-α-BTX/Streptavidin beads and probed for Rapsn and actin (Figure 5A). As shown in Figure 5B, the amount of Rapsn and actin associated with surface AChRs was similar between WT and N88K mt myotubes. When co-expressed in HEK293 cells, AChR subunits precipitated by WT or N88K mt Rapsn were similar (Figure 5—figure supplement 1A and B), indicating the mutation has little effect on the binding to AChRs. Actin-anchored Rapsn-AChR complexes are resistant to low concentration of detergents (Moransard et al., 2003). We found that the amount of Rapsn that could be solubilized by Triton X-100 was similar between N88K mt and WT myotubes (Figure 5—figure supplement 1C and D). These results suggest that the N88K mutation may not impair Rapsn association with actin cytoskeleton components.

Figure 5 with 1 supplement see all
Reduced E3 ligase activity in N88K mt Rapsn.

(A) Schematic diagram of extraction of surface AChRs from C2C12 myotubes. Live C2C12 myotubes were incubated with biotin-α-BTX at 4°C for 1 hr to capture AChR complex, and then were lysed. Resulting biotin-α-BTX-AChR complex in lysates were precipitated by streptavidin-coupled agarose beads. Cell lysates, precipitated AChRs and AChR-associated proteins were examined by western blotting. (B) Comparable amounts of Actin and Rapsn were co-precipitated by surface AChR between WT and N88K mt C2C12 myotubes. Surface AChRs from WT or N88K mt myotubes were isolated, and associated Rapsn and actin were examined by western blotting. (C) Reduced Rapsn E3 ligase activity by N88K mutation in HEK293T cells. HEK293T cells were transfected with HA tagged WT, C366A, or N88K mt Rapsn, along with Flag-δ-AChR and Nedd8-Myc. After 48 hr, cells were lysed and precipitated with anti-Flag beads to pull down δ-AChR. The precipitated δ-AChR was blotted with anti-Nedd8 antibody to examine its neddylation. (D) Quantitative data of neddylated δ-AChR in (C) (mean ± SEM), ***, p<0.001, One-way ANOVA, n = 3. (E–H) Reduced Rapsn E3 ligase activity in N88K mt cultured myotubes and in mt mice. (E, F) WT and N88K mt cultured myotubes were treated with Agrin for 2 hr. Myotubes were lysed and precipitated with anti-Nedd8 antibody. The resulting lysates and precipitated proteins were blotted with indicated antibodies to reveal neddylated δ-AChR, readout for E3 ligase activity of Rapsn (E). (F) Quantitative data in (E) (mean ± SEM), ***, p<0.001, unpaired t-test, n = 3. (G) Neddylated δ-AChR was examined in WT or N88K mt mice. (H) Quantitative data in (G) (mean ± SEM), ***, p<0.001, unpaired t-test, n = 3. Also see Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.49180.016

Our recent study revealed that Rapsn possesses E3 ligase activity, which is required for AChR clustering and NMJ formation (Li et al., 2016). In a working model, Rapsn increases AChR clusters by enhancing AChR neddylation, a posttranslational modification with Nedd8 (an Ub-like molecule) (Li et al., 2016; Li et al., 2018). To examine whether N88K mutation impairs Rapsn-mediated neddylation of AChRs, HA-tagged WT or N88K mt Rapsn was co-transfected with Flag-tagged δ-AChR and Myc-tagged Nedd8 into HEK293T cells. δ-AChR was purified by anti-Flag antibody and probed with anti-Nedd8 antibody. Nedd8-conjugated δ-AChR was apparent in HEK293T cells co-expressing WT Rapsn (Figure 5C and D); however, Nedd8 signaling was dramatically reduced in cells co-expressing N88K Rapsn (Figure 5C and D), to a level of cells expressing C366A, an E3 ligase-dead Rapsn mt (Li et al., 2016). These results suggest that N88K mutation may impair the E3 ligase activity of Rapsn. To further test this hypothesis, we compared neddylated δ-AChR level between WT and N88K mt C2C12 myotubes and found that neddylated δ-AChR in N88K mt myotubes was remarkably reduced, compared with WT controls (Figure 5E and F). Finally, we examined neddylated δ-AChR level in skeletal muscles of WT and N88K mt mice. As shown in Figure 5G and H, neddylated δ-AChR level was reduced in muscles of N88K mt mice, compared with WT controls. Together these results suggest that impaired E3 ligase activity may be a mechanism of N88K mutation.

Reduced Rapsn tyrosine phosphorylation by N88K mutation

Agrin binds to LRP4 to stimulate MuSK to induce AChR clustering. How signal is transduced from MuSK to Rapsn was not well understood. Rapsn was tyrosine phosphorylated in electric organs of Torpedo California where AChRs are enriched (Mohamed and Swope, 1999) and in heterologous cells when expressed alone or together with MuSK (Lee et al., 2008). We characterized Rapsn tyrosine phosphorylation in muscle cells in response to Agrin treatment. Rapsn and MuSK were precipitated with respective antibodies from C2C12 myotubes treated with Agrin for different times and probed with anti-p-Tyr antibody. As shown in Figure 6A, MuSK activation occurred within 10 min, which was followed by Rapsn tyrosine phosphorylation. On the other hand, the increased in δ-AChR neddylation, an indicator of Rapsn E3 ligase activity (Li et al., 2016), was not peaked until 90 min after Agrin stimulation (Figure 6B). These data, quantified in Figure 7J, suggest that these events occur sequentially.

Impaired Y86 phosphorylation by N88K mutation.

(A) Agrin treatment induced tyrosine phosphorylation of Rapsn in cultured myotubes. WT cultured myotubes were treated with Agrin for indicated times. Cells were lysed and were incubated with anti-Rapsn antibody to precipitate Rapsn protein, and probed with anti-p-Tyr antibody to examine tyrosine phosphorylation of Rapsn. MuSK tyrosine phosphorylation was examined as positive controls. (B) Agrin treatment induced E3 ligase activity of Rapsn in culture myotubes. WT cultured myotubes were treated with Agrin for indicated times. Neddylated δ-AChR was examined to reveal E3 ligase activity of Rapsn. (C) Reduced tyrosine phosphorylation of Rapsn by N88K mutation in cultured myotubes. WT, N88K mt, Rapsn null mt (-/-) cultured myotubes were treated with Agrin for 2 hr, and then tyrosine phosphorylation of Rapsn was examined. (D) Quantitative data in (C) (mean ± SEM), *, p<0.05, unpaired t-test, n = 3. (E–G) Reduced tyrosine phosphorylation of Rapsn by N88K mutation in mt mice. Rapsn protein was precipitated with anti-Rapsn antibody from muscle homogenates of WT or N88K mice at E14 (E) and P0 (F) and probed with anti-p-Tyr antibody to examine tyrosine phosphorylation of Rapsn. (G) Quantitative data in (E) and (F) (mean ± SEM), **, p<0.01, ***, p<0.001, unpaired t-test, n = 3. (H) Reduced MuSK-induced Rapsn tyrosine phosphorylation by N88K and Y86F mutations in HEK293T cells. HEK293T cells were transfected HA tagged WT or indicated mt Rapsn, along with Flag tagged MuSK, or not. Precipitated Rapsn protein with anti-HA beads was probed with anti-p-Tyr antibody to reveal tyrosine phosphorylation of Rapsn. (I) Quantitative data of (H) (mean ± SEM), *, p<0.05, **, p<0.001, unpaired t-test, n = 3. (J) Abolished MuSK-induced TPR1-7 (TPR) tyrosine phosphorylation by N88K and Y86F mutations in HEK293T cells. Same as (H), MuSK-induced WT, Y86F, or N88K mt TPR was detected in HEK293T cells.

https://doi.org/10.7554/eLife.49180.020
Figure 7 with 1 supplement see all
Critical roles of Y86 phosphorylation in activating Rapsn E3 ligase activity and AChR clustering.

(A) Reduced E3 ligase activity of Rapsn by Y86F mutation in transfected HEK293T cells. (B) Quantitative data of neddylated δ-AChR in (A) (mean ± SEM), ***, p<0.001, One-way ANOVA, n = 3. (C) Reduced Rapsn tyrosine phosphorylation in Y86F mt myotubes, compared with WT controls. (D) Quantitative data of tyrosine phosphorylation of Rapsn in (C) (mean ± SEM), *, p<0.05, unpaired t-test, n = 3. (E) Fewer Agrin-induced AChR clusters in Y86F mt C2C12 myotubes, compared with WT controls. Arrows, AChR clusters. (F) Quantitative data in (E), (mean ± SEM), **, p<0.01, ***, p<0.001, Two-way ANOVA, n = 20 cells. (G) Reduced E3 ligase activity of Rapsn in Y86F mt myotubes. (H) Quantitative data in (G) (mean ± SEM), ***, p<0.001, unpaired t-test, n = 3. (I) Agrin treatment increased Rapsn self-association. Cultured C2C12 cells were transfected with HA- and EGFP-tagged Rapsn, respectively. Resulting myotubes were treated with Agrin. Cell lysates were subjected to co-immunoprecipitation with anti-HA beads, and probed with anti-GFP antibody to reveal the association between Rapsn-HA and Rapsn-EGFP. (J) Quantitative analysis of MuSK and Rapsn tyrosine phosphorylation in Figure 6A, neddylated δ-AChR in Figure 6B, and Rapsn self-association in Figure 7I. Data were shown as mean ± SEM, **, p<0.01, ***, p<0.001 (compared with time 0), One-way ANOVA, n = 3. (K–N) Reduced Rapsn self-association by Y86F (K) or N88K (M) mutation revealed by co-immunoprecipitation in HEK293T cells. Quantitative data of WT or Y86F Rapsn self-association(L); WT or N88K Rapsn self-association (M) (mean ± SEM), *, p<0.05; ***, p<0.001, unpaired t-test, n = 3. Also see Figure 7—figure supplement 1.

https://doi.org/10.7554/eLife.49180.022

To determine whether N88K mutation alters Rapsn tyrosine phosphorylation in muscle cells, we studied N88K mt C2C12 myotubes together with WT and Rapsn null (-/-) C2C12 myotubes. Compared with WT C2C12 myotubes, Agrin-stimulated Rapsn tyrosine phosphorylation in N88K mt myotubes was dramatically reduced (Figure 6C and D). These results demonstrate that Rapsn becomes tyrosine phosphorylated in response to Agrin stimulation and this event is inhibited by N88K mutation. To determine whether N88K mutation alters Rapsn tyrosine phosphorylation in vivo, Rapsn protein was precipitated with anti-Rapsn antibody from muscle homogenates of mice at E14 and P0 and probed with anti-p-Tyr antibody (Figure 6E–G). Rapsn tyrosine phosphorylation from N88K mt mice was reduced compared with WT muscles. These results demonstrate that in vivo tyrosine phosphorylated of Rapsn was reduced in N88K mt mice.

To determine whether tyrosine phosphorylation is critical for Rapsn’s function, we sought to identify the tyrosine residues that are phosphorylated upon MuSK activation. HA-tagged Rapsn was co-transfected with Flag-tagged MuSK in HEK293T cells, which induced tyrosine phosphorylation of Rapsn (Figure 6H), consistent with previous work (Lee et al., 2008). Tyrosine phosphorylation was reduced in N88K mt Rapsn (Figure 6H and I), as observed in aforementioned studies with C2C12 myotubes (Figure 6C and D). We focused on tyrosine residues immediately flanking N88: Y59 in TPR2, Y86 and Y105 in TPR3, and mutated them individually to phenylalanine (F). Tyrosine phosphorylation of Y59F and Y105F Rapsn was similar to WT Rapsn in HEK293T cells co-expressing MuSK (Figure 6H and I), suggesting that these two residues may not be the target residue. However, Y86F mutation dramatically reduced tyrosine phosphorylation of Rapsn (Figure 6H and I). To test this notion further, we generated a recombinant protein containing only TPR domain. As shown in Figure 6J, MuSK-mediated tyrosine phosphorylation was completely abolished by the Y86F as well as N88K mutation. Together, these results demonstrate that Y86 may be a site in Rapsn that becomes tyrosine phosphorylated upon MuSK activation, and Y86 phosphorylation is regulated by N88K mutation.

Y86 phosphorylation for E3 ligase activity and AChR clustering

To investigate the role of Y86 phosphorylation, we determined whether its mutation impacts on E3 ligase activity of Rapsn. HEK293T cells were transfected with Flag-δ-AChR, Myc-tagged Nedd8 and HA-tagged WT, Y86F, or C366A Rapsn and tested δ-AChR neddylation as described in Figure 5C. δ-AChR neddylation was reduced in cells expressing Y86F or C366A (Figure 7A and B), compared with WT control, suggesting that Y86F mutation impairs the E3 ligase activity of Rapsn. To examine the role of Y86 phosphorylation on AChR cluster formation, we generated Y86F mt C2C12 cells by CRISPR-Cas9 (Figure 7—figure supplement 1A). The mutation was confirmed by genomic DNA sequencing (Figure 7—figure supplement 1B). Y86F mutation did not alter mRNA or protein levels of Rapsn (Figure 7—figure supplement 1C, and Figure 7C) or Rapsn protein stability (Figure 7—figure supplement 1D and E). Agrin-induced tyrosine phosphorylation was reduced, but not abolished in Y86F mt C2C12 myotubes (Figure 7C and D), consistent with results of HEK293T cells (Figure 6H). Remarkably, Agrin-induced clusters were fewer in Y86F mt C2C12 myotubes, compared with WT control (Figure 7E and F), indicating a necessary role of Y86 phosphorylation in Agrin-mediated AChR clustering. In support of this notion, δ-AChR neddylation was reduced in Y86F mt C2C12 myotubes, compared with WT control (Figure 7G and H).

Next, we investigated how Y86F mutation impairs E3 ligase activity of Rapsn. E3 ligases including those containing RING-domain are regulated by self-association (Bian et al., 2017; Ho et al., 2015; Koliopoulos et al., 2016; Liew et al., 2010; Metzger et al., 2014; ; Nikolay et al., 2004). The TPR domains in the N-terminus of Rapsn are thought to mediate self-association and thus form aggregates in heterologous cells (Maimone and Merlie, 1993; Ramarao et al., 2001). We posited that tyrosine phosphorylation of Rapsn may promote self-association and thus activates E3 ligase activity. To test this, C2C12 myoblasts were co-transfected with Rapsn that were tagged by EGFP and HA, respectively (Figure 7I). Resulting myotubes were treated with Agrin and examined for self-binding by co-precipitation. Agrin increased the amount of Rapsn-EGFP co-precipitated by Rapsn-HA, plateaued ~90 min of Agrin treatment. As summarized in Figure 7J, upon Agrin stimulation, tyrosine phosphorylation of MuSK occurs prior to that of Rapsn; and subsequently, Rapsn self-association and δ-AChR neddylation were increased, suggesting E3 ligase activity might be regulated by self-association. To examine this, we investigated whether Y86F alters Rapsn self-association. HEK293T cells were transfected with EGFP-tagged and HA-tagged Rapsn. Lysates were incubated with beads immobilized with anti-HA antibody to purify HA-tagged Rapsn and probed with anti-GFP antibody. Compared with WT control, less amount of EGFP-tagged Y86F mt Rapsn was co-precipitated by HA-tagged Y86F Rapsn (Figure 7K and L), suggesting impaired ability of mt Rapsn to self-associate. In agreement, self-association of N88K Rapsn was also reduced, compared with WT Rapsn (Figure 7M and N), suggesting that Y86 and N88 are necessary for Rapsn self-association. Together, these results support a working model that MuSK stimulates AChR cluster formation by increasing Rapsn phosphorylation and self-association and thus enhancing its E3 ligase activity.

Failure of Y86F-Rapsn in rescuing NMJ deficits in N88K mt mice

To investigate the role of Y86 phosphorylation in vivo, we generated adeno-associated virus (AAV, AAV-PHP.B) expressing WT Rapsn (AAV-WT-EGFP) or Y86F mt Rapsn (AAV-Y86F-EGFP) (Figure 8—figure supplement 1A and B). Equal volume (10 μl) of AAV viruses in same titer (1.3 × 1013 vg/ml) were intramuscularly injected into N88K mt thigh muscles of embryos at E13. Thigh muscles were isolated at P0 and subjected to morphological characterization. As shown in Figure 8—figure supplement 1C, muscles infected with AAV were visible for EGFP. AChR clusters in N88K thigh muscles were reduced in number and appeared to be thin, compared with WT controls (Figure 8A, white arrows), as observed in diaphragm muscles (Figure 1C and D). Remarkably, AChR clusters were readily detectable in muscles of N88K mt mice that were injected with AAV-WT-EGFP (Figure 8A). Unlike AChR clusters in N88K mt mice that were elongated, oval-shaped clusters were detectable in AAV-WT-EGFP-infected N88K mt thigh muscles. Also increased were the size and fluorescence intensity of AChR clusters (Figure 8A–D). These results demonstrate that N88K mutation-caused NMJ impairment could be attenuated by viral expression of WT Rapsn. Noticeably, the rescue effects were not observed in mice injected with AAV-Y86F-EGFP (Figure 8A–D). These results provide in vivo evidence that Y86 is critical to Rapsn’s function in NMJ formation.

Figure 8 with 1 supplement see all
Rescue of NMJ deficits by WT Rapsn but not phospho-preventing Y86F mt.

(A) Thigh muscles of N88K mt were infected with AAV-WT-Rapsn-EGFP (N88K + AAV-WT-EGFP), or infected with AAV-Y86F-Rapsn-EGFP (N88K + AAV-Y86F-EGFP) at E13. The resulting P0 muscles were stained with Flour 594-α-BTX (red) and anti-NF/Syn antibodies (blue) to examine AChR clusters, using uninfected WT or N88K mt as positive or negative controls. (B–D) Quantitative analysis of AChR cluster fluorescence intensity (B), AChR cluster size (C), AChR cluster length (D). White arrows, AChR clusters. Data were shown as mean ± SEM; *, p<0.05; ***, p<0.001, ns, no significant difference, One-way ANOVA, n = 3. Also see Figure 8—figure supplement 1.

https://doi.org/10.7554/eLife.49180.026

Discussion

This study provides evidence that the N88K mutation prevented muscle fibers from forming aneural AChR clusters prior to innervation as well as nerve-induced AChR clusters. Axon terminals were extensively arborized in N88K mt mice, with increased secondary and tertiary nerve branches and reduced density of synaptic vesicles phenotypes observed in mt mice lacking LRP4 or MuSK (DeChiara et al., 1996; Weatherbee et al., 2006; Wu et al., 2012b). These results uncover previously unappreciated impacts of N88K mutation on the NMJ and contribute to a better understanding of pathological mechanisms of how N88K mutation causes CMS. At the molecular level, studies of N88K mutation led to discover a pathway by which signal is transduced from MuSK to Rapsn. Agrin is a factor utilized by motoneurons to direct NMJ formation, in particular postsynaptic differentiation (McMahan, 1990). It binds to LRP4 and thus activates MuSK (Kim et al., 2008; Zhang et al., 2008; Zong et al., 2012). Like Rapsn, Agrin, LRP4, and MuSK are absolutely necessary for NMJ formation (DeChiara et al., 1996; Gautam et al., 1996; Gautam et al., 1995; Weatherbee et al., 2006). However, signaling events downstream of MuSK were unclear except that Dok7 is a necessary adapter protein that is believed to dimerize MuSK (Okada et al., 2006); and Rapsn that could function as an adapter protein to bridge AChRs to the cytoskeleton and/or as an E3 ligase to promote neddylation of the AChR (Li et al., 2016; Li et al., 2018). Although tyrosine phosphorylation of Rapsn has been observed in torpedo electric organs (Mohamed and Swope, 1999) and in MuSK- and Rapsn-cotransfected cells (Lee et al., 2008), its functional significance was unclear. The N88K mutation seemed to have little effect on the levels of Rapsn or AChR in muscle cells. We demonstrate that it may alter the phosphorylation of Y86, an adjacent tyrosine residue that is required for Agrin signaling. First, Y86 became tyrosine phosphorylated upon Agrin stimulation. Second, mutating Y86 to F, to block tyrosine phosphorylation, reduced MuSK-dependent tyrosine phosphorylation of full-length Rapsn, and abolished MuSK-dependent tyrosine phosphorylation of TPR1-7 of Rapsn, suggesting Y86 is a major tyrosine-phosphorylation site at TPR domain. Third, in HEK293T cells, Y86F mutation reduced Rapsn E3 ligase activity to comparable, minimal level as Rapsn C366A mt whose E3 ligase activity was abolished (Li et al., 2016). In agreement, E3 ligase activity of Rapsn was reduced in Y86F knock-in myotubes, suggesting a necessary role of Y86 phosphorylation in E3 ligase activity regulation. Fourth, WT Rapsn was able to rescue AChR clustering deficits in N88K mt mice; this rescue effect was not observed with Y86F mt Rapsn. Finally, we demonstrate that N88 and Y86 are both necessary for Rapsn self-association, a process that has been shown to be necessary for E3 ligase activation (Bian et al., 2017; Ho et al., 2015; Koliopoulos et al., 2016; Liew et al., 2010; Metzger et al., 2014; Nikolay et al., 2004; ). A parsimonious explanation of these results is that MuSK activation causes, directly or indirectly, Rapsn tyrosine phosphorylation at Y86, which promotes Rapsn self-association and thus activate the E3 ligase activity, revealing a mechanism for Rapsn activation. These results also suggest a novel, molecular pathological mechanism by which N88K mutation impairs the NMJ formation and maintenance.

Rapsn has 14 tyrosine residues, including 12 in the TPR domain. Mutation of the Y86 abolished MuSK-dependent tyrosine phosphorylation of the TPR domain, suggesting that Y86 is a key tyrosine phosphorylation site in this region or its mutation prevents others tyrosine residues from being phosphorylated. Interestingly, tyrosine phosphorylation of WT Rapsn was reduced, but not abolished by the Y86F mutation. About 40% of MuSK-dependent tyrosine phosphorylation remained in Y86F mt. These results could suggest that tyrosine residues in C-terminal regions of Rapsn may be a target for MuSK regulation. This region contains a coiled-coil domain that could bind to the AChR (Ramarao and Cohen, 1998), and a RING-H2 domain that could bind to β-dystroglycan (Bartoli et al., 2001) and possess the E3 ligase activity (Li et al., 2016). It would be interesting to determine whether these activities are regulated by tyrosine phosphorylation. Consistent with this notion, Agrin treatment can enhance the AChR-Rapsn interaction (Luo et al., 2008; Moransard et al., 2003) and the association of the AChR-Rapsn complex to cytoskeleton (Moransard et al., 2003).

Both MuSK kinase activity and Rapsn are necessary for prepatterning or the formation of aneural AChR clusters (Lin et al., 2001; Yang et al., 2001b); and Rapsn is known to interact with MuSK in the absence of Agrin (Apel et al., 1997). Further, LRP4 could interact with MuSK and thus maintain its activity in the absence of Agrin (Kim et al., 2008; Zhang et al., 2008). Indeed, Rapsn phosphorylation could be detectable at E14, when aneural AChR clusters are yet to be innervated by axons. Together, these observations suggest that tyrosine phosphorylation of Rapsn could contribute to the formation of aneural AChR clusters or muscle prepatterning. It is worthy pointing out that evidence is lacking whether Rapsn is a direct substrate protein of MuSK.

Pathological mechanisms of the N88K mutation have been examined previously. It was shown to reduce Rapsn’s ability to induce AChR clusters in HEK and TE671 cells (Cossins et al., 2006; Ohno et al., 2002). In agreement, the N88K mt was shown here to reduce Rapsn’s ability to induce AChR clusters in HEK293T cells (Figure 4A). In N88K knock-in mt mice and in CRISPR/Cas9-generated C2C12 myotubes, AChR clusters were almost diminished. However, N88K seemed to be able to induce AChR clusters in Rapsn mt myotubes (Cossins et al., 2006). The cause of this discrepancy is unclear. In the current study, the impact of the N88K mutation was inferred mainly by loss-of-function approaches, both in vitro and in vivo, without changing endogenous Rapsn level (Figure 1 and Figure 4E and F). Rapsn is known to induce AChR clusters at low concentrations, but inhibit AChR clusters at high level (Han et al., 1999; Yoshihara and Hall, 1993). This inverted U-function could complicate the interpretation of results of overexpressing WT or mt Rapsn.

CMS patients carrying the N88K mutation show variable phenotypes, from serious, early-onset symptoms to mild, late-onset symptoms (Cossins et al., 2006; Milone et al., 2009; Müller et al., 2003), even in patients with homozygous N88K mutation. In severe cases, N88K heteroallelic with c.966 + 1GT, L14P, or a frameshift mutation may cause postnatal death (Maselli et al., 2003; Milone et al., 2009; Richard et al., 2003), indicating N88K mutation could have seriously detrimental effects on NMJ formation or maintenance in human subjects. Overall, the symptoms of CMS patients with homozygous N88K mutation are milder than that of patients carrying heteroallelic N88K mutation with a second mutation. These secondary mutations including sequence deletions or insertions, such as c.1177-1178del AA, p.V50-S55del, p.K373del, and c.553-554insGTTCT, are often more disruptive (Milone et al., 2009). We also note that phenotypes of N88K mt mice seem to be more severe than symptoms of CMS patients. Almost all N88K mt mice died within 24 hr of birth. A simple explanation of these observations may be genetic difference. Gene mutations identified in patients with various diseases such as Lesch-Nyhan syndrome, Lowe syndrome, galactosemia diseases often failed to reproduce clinic symptoms in mouse models (Elsea and Lucas, 2002). On the other hand, a knock-in mutation of AQP2, a gene implicated in hereditary non-X-linked nephrogenic diabetes insipidus, causes more severe phenotypes in mice than in human subjects (Yang et al., 2001a). Even in same species, effects of a mutation could have variable impacts on different strains of mice, for example, the Agrin mutation, Agrinnm380 (Bogdanik and Burgess, 2011). Furthermore, it is possible that some patients with the N88K mutation are unable to survive and thus were not included in clinic reports.

In sum, this study demonstrates that the most prevalent CMS-associated mutation in Rapsn is causal to NMJ deficits. Not only does it reveal pathological mechanisms of how the N88K mutation alters NMJ development, the study also uncovers molecular mechanisms by which Rapsn is regulated upon MuSK activation. The results support a model where Rapsn becomes phosphorylated on tyrosine residues for self-association and E3 ligase activity, which are necessary for AChR clustering. Intriguingly, NMJ deficits in N88K mt mice could be diminished by a virus expressing WT Rapsn, suggesting that gene therapy may be beneficial for CMS.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent
(M. musculus)
Rapsn-/-PMID: 7675108
Genetic reagent
(M. musculus)
N88KThis paperSee detail information in Materials and methods .
Cell line (Homo sapiens)HEK293TATCCCat#:CRL-3216
RRID: CVCL_0042
From ATCC; Cell identity has been confirmed by STR profiling and cell line was found to be free of Mycoplasma.
Cell line (M. musculus)C2C12ATCCCat#: CRL-1772
RRID: CVCL_0188
From ATCC; Cell identity has been confirmed by STR profiling and cell line was found to be free of Mycoplasma.
Cell line (M. musculus)N88K mt C2C12This paperSee detail information in Material and methods).
Cell line (M. musculus)Y86F mt C2C12This paperSee detail information in Material and methods.
Cell line (M. musculus)Rapsn-/- C2C12, clone 11–7PMID: 10414969
AntibodyMouse monoclonal anti-HA AgaroseThermo Fisher ScientificCat#: 26181,
RRID: AB_2537081
1: 40 for IP
AntibodyGoat anti-rabbit IgG conjugated with Alexa Fluor 488Thermo Fisher ScientificCat#: A-11008
RRID: AB_10563748
IHC (1:500)
AntibodyRabbit polyclonal anti-ActinCell Signaling TechnologyCat #: 4967,
RRID: AB_330288
WB (1:10000)
AntibodyRabbit polyclonal anti- α-ActinAbcamCat #: ab52218
RRID: AB_870573
WB (1:1000)
AntibodyRabbit polyclonal anti-FlagSigma-AldrichCat #: F7425
RRID: AB_439687
WB (1:1000)
AntibodyMouse monoclonal anti-GAPDH (Clone, 6C5)Santa Cruz BiotechnologyCat #: sc-32233,
RRID: AB_627679
WB (1:10000)
AntibodyRabbit polyclonal anti-neurofilamentCell Signaling TechnologyCat#: 2837,
RRID: AB_823575
IHC (1:1000)
AntibodyRabbit polyclonal anti-synapsinCell Signaling TechnologyCat#: 5297,
RRID: AB_2616578
IHC (1:1000)
AntibodyMouse monoclonal anti-Ubiquitin (Clone, Ubi-1)AbcamCat#: ab7254,
RRID: AB_305802
WB (1:1000)
AntibodyRabbit polyclonal anti-GFPCell Signaling TechnologyCat#: 2555,
RRID: AB_10692764
WB (1:1000)
AntibodyMouse monoclonal P-Tyr-100Cell Signaling TechnologyCat #: 9411
RRID: AB_331228
WB (1:1000)
AntibodyMouse monoclonal Anti-Flag affinity gel (Clone, M2)Sigma-AldrichCat#: A2220,
RRID: AB_10063035
1: 50 for IP
AntibodyRabbit polyclonal anti-HASigma-AldrichCat#: H6908,
RRID: AB_260070
WB (1:2000)
AntibodyMouse monoclonal anti-Rapsn (Clone 1234)AbcamCat#: ab11423,
RRID: AB_298028
WB (1:1000)
AntibodyRabbit polyclonal anti-TransferrinAbcamCat#: ab82411,
RRID: AB_1659060
WB (1:1000)
AntibodyMouse monoclonal anti-δ-AChR (Clone, 88B)Thermo Fisher ScientificCat#: MA3-043,
RRID: AB_2081037
WB (1:1000)
AntibodyRabbit polyclonal anti-RapsnPMID: 18940591WB (1:1000)
AntibodyGoat polyclonal anti-α-AChRPMID: 3484485WB (1:1000)
AntibodyRabbit polyclonal anti-β-AChRPMID: 3484485WB (1:1000)
AntibodyHorseradish peroxidase (HRP)-conjugated goat anti-rabbit IgGThermo Fisher ScientificCat#: 32260,
RRID: AB_1965959
WB (1:5000)
AntibodyHorseradish peroxidase (HRP)-conjugated goat anti-Mouse IgGThermo Fisher ScientificCat#: 32230,
RRID: AB_1965958
WB (1:5000)
AntibodyHorseradish peroxidase (HRP)-conjugated goat anti-Rat IgGThermo Fisher ScientificCat#: 31470,
RRID: AB_228356
WB (1:5000)
Recombinant ProteinAgrinR and D SystemsCat#: 550-AG-10050 ng / ml for induction of AChR clusters in culture myotubes
OtherImmunofluorescence of NMJ in diaphragm and musclesPMID: 18278041
PMID: 22794264
OtherElectron microscopic analysis
OtherGeneration of Gene-modified C2C12 cellsPMID: 27839998
OtherIsolation of cell surface proteinPMID: 22157653
OtherIsolation of surface AChR and their associated proteinsPMID: 18940591
OtherAAV Virus productionPMID: 30626963 and
Information in Addgene: https://www.addgene.org/protocols/aav-production-hek293-cells/

Mice

Rapsn null (-/-) mt mice were a kind gift from Dr. Peter Noakes (Gautam et al., 1995). The N88K mt mice were generated by the University of Rochester Mouse Genome Editing Resource using CRISPR/Cas9 approach, as described previously (Li et al., 2016). Briefly, a single strand DNA template (ssDNA), CCGTG GTCCA GATTG ATACT GCTCG GGGAC TGGAG GATGC TGACT TCCTG CTCGA AAGCT ACCTC AAGTT AGCTC GCAGC AATGA GAAGC TATGT GAGTT CCACA AAACC ATCTC CTACT GCAAG ACCTG CCTCG G, containing the desired mutations was synthesized and purified by Integrated DNA Technologies. SgRNA was generated by a MEGAshortscript T7 kit (Life Technologies) using template sequence (GCTCG AAAGC TACCT GAACC) cloned in pX330 plasmid (Addgene #42230) and was purified using MEGAclear kit (Life Technologies). Mixture of Cas9 mRNA (TriLink Biotechnologies, 100 ng / μl), sgRNA (50 ng / μl), and ssDNA (100 ng / μl) was injected into fertilized eggs from C57BL/6J mice (Jackson Laboratory, Stock #000664). Viable two-cell stage embryos were transferred to pseudo-pregnant ICR females to generate founder mice, which were subsequently bred with C57BL/6J mice for germline transmission to generate F1 mice. Mice carrying N88K mutation were screened by PCR analysis and confirmed by DNA sequencing. Primers: 5’- GCTCG AAAGC TACCTG AACCT GGCG-3’ and 5’-CACGA GGTTCT CAGGG AGCCT CA-3’ were used to verify WT genomic DNA, and primers: 5’-CTTCC TGCTC GAAAG CTACC TCAAG TTAGC T-3’ and 5’-CACGA GGTTC TCAGG GAGCC TCA-3’ were used to verify N88K mt genomic DNA. C57BL/6 mice were used as WT controls. Mice were housed in cages in a room with 12 hr light-dark cycle with ad libitum access to water and rodent chow diet (Diet P3000). Embryo and P0 pups of either sex were analyzed, unless otherwise indicated. Animal protocols have been approved by the Institutional Animal Care and Use Committee of Case Western Reserve University.

Reagents and antibodies

Request a detailed protocol

Following reagents were purchased from Thermo Fisher Scientific (Waltham, MA): Flour-594 conjugated α-Bungarotoxin (Flour 594-α-BTX; Catalog number, B-13423; 1:1000 for staining), Biotin conjugated α-Bungarotoxin (Biotin-α-BTX, Catalog number, B1196), Sulfo-NHS-SS-Biotin (Catalog number, PG82077), streptavidin-coupled agarose beads (Catalog number, 20349), goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Catalog number, A-11008; 1: 500 for staining), mouse anti-HA agarose (Catalog number, 26181). Mouse anti-GAPDH (Catalog number, NB 600–501; 1:10000 for WB) was from Novus (Littleton, CA). Rabbit anti-Actin (Catalog number, 4967; 1: 5000 for western blotting), rabbit anti-Nedd8 (Catalog number, 10695299; 1:1000 for WB and IP), rabbit anti-neurofilament (Catalog number, 2837S; C28E10, 1:1000 for staining), rabbit anti-synapsin (Catalog number, 5297; D12G5, 1:1000 for staining) and mouse anti-phospho-tyrosine (Catalog number, 9411S; P-Tyr-100; 1:1000 for western blotting) were from Cell signaling technology (Boston, MA). Cycloheximide (CHX, Catalog number, C7658-5G; 50 μg / ml), rabbit anti-HA (Catalog number, H6908; 1:1000 for western blotting), rabbit anti-Flag (Catalog number, F7425; 1:1000 for western blotting), HA peptide (Catalog number, 11666975001), anti-Flag M2 affinity gel (Catalog number, A2220) were from Sigma (Mendota Heights, MN). Mouse anti-Ubiquitin (Catalog number, ab7254; 1:1000 for western blotting), rabbit anti-α-Actin (Catalog number, ab52218; 1: 10000 for western blotting), mouse anti-Rapsn (Catalog number, ab11423; clone 1234, 1:1000 for western blotting), rabbit anti-Transferrin (Catalog number, ab82411; 1: 1000 for western blotting) were from Abcam (Cambridge, MA). Mouse anti-δ-AChR (C4; 1:1000 for western blotting) was from Santa Cruz Biotechnology (Dallas, TX). Rabbit anti-Rapsn (2741), goat anti-α-AChR and rabbit anti-β-AChR antibodies were described previously (Barik et al., 2014; Luo et al., 2008; Wu et al., 2012a; Zhao et al., 2017). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Catalog number, 32260), goat anti-mouse IgG (Catalog number, 32230), goat anti-rat IgG (Catalog number, 31470) antibodies (1:5000 for western blotting) were from Pierce (Rockford, IL).

Immunofluorescence

Request a detailed protocol

The procedures for staining muscles or diaphragms were described previously (Li et al., 2016; Li et al., 2008; Wu et al., 2012b). Briefly, skeletal muscles or diaphragms were fixed in 4% paraformaldehyde in PBS for 24 hr, and then dissected in PBS. Dissected samples were rinsed with 0.1 M glycine in phosphate-buffered saline (PBS) for 1 hr at room temperature and followed by three-time washing in PBS. Samples were then incubated with the blocking buffer (5% BSA, 5% goat serum, 1% Triton X-100 in PBS) for 1 hr at room temperature and incubated with primary antibodies in blocking buffer at 4°C overnight. Next day, after washing three times with washing buffer (1% Triton X-100 in PBS), samples were incubated with fluorescent-labeled secondary antibodies at room temperature for 1–2 hr. Samples were then washed with washing buffer and mounted with Vectashield mounting medium (H1000, Vector Laboratories, Burlingame, CA) and coverslip. Images were collected with a Zeiss confocal laser scanning microscope (LSM 800) and collapsed into a single image. AChR clusters and axon branches in left, ventral diaphragms were quantified within 1 mm of primary phrenic nerve branches.

Western blot analysis

Request a detailed protocol

Western blot was performed as described previously (Barik et al., 2014; Zhao et al., 2017).

Electron microscopic analysis

Request a detailed protocol

The procedures for electron microscopic were described previously (Li et al., 2016; Wu et al., 2015; Wu et al., 2012b).

Electrophysiological recording

Request a detailed protocol

The procedures for electrophysiological recording of P0 diaphragms were described as previously (Li et al., 2008; Shen et al., 2018).

C2C12 culture and generation of Gene-modified C2C12 cells

View detailed protocol

Mouse myoblasts C2C12 were purchased from ATCC (Catalog number, CRL-1772, Manassas, VA). Rapsn null mt muscle cells (clone 11–7) were kindly provided by Dr. C. Fuhrer. The procedures for C2C12 culture and generation of knock-in mt C2C12 cell by CRISPR-Cas9 were described previously (Li et al., 2016; Zhang et al., 2012; Zhang et al., 2007). The sgRNA carrying targeting sequence GCTCG AAAGC TACCT GAACC was used for generation of N88K mt C2C12; sgRNA carrying targeting sequence CAT TGC TGC GCG CCA GGT TC was used for generation of Y86F mt C2C12. For comparing Rapsn tyrosine phosphorylation and E3 ligase activity between WT and N88K mt culture myotubes, myotubes were treated with Agrin for 2 hr, because Agrin activates MuSK within 10 min of stimulation and tyrosine phosphorylation of Rapsn that plateaus around 20 min (Figure 6A), and Agrin induces the formation of AChR clusters beginning around 2 hr of stimulation (Ngo et al., 2012). For studying Rapsn self-association in culture myotubes (Figure 7I), overexpression of high dosage of Rapsn could induce Rapsn aggregation in the absence of Agrin (data not shown), several dosages of Rapsn vectors were tested, and 3.5 μg Rapsn-HA vector and 3.5 μg Rapsn-EGFP vector were used to transfect 10 cm culture plate. At these dosages, through viewing GFP fluorescence, we could see expression of Rapsn at culture myotubes, but these myotubes didn’t have obvious Rapsn aggregates.

Isolation of cell surface protein

Request a detailed protocol

The procedures for isolating surface proteins were described previously (Chen et al., 2012). Briefly, cells were washed twice with ice-cold PBS followed by incubation with 10 ml 0.25 mg/mL Sulfo-NHS-SS-Biotin in PBS containing Mg2+/Ca2+ at 4°C for 45 min. Reaction was quenched by adding 10 mM Glycine. Cells were lysed in RIPA buffer containing 150 mM NaCl, 25 mM Tris-HCl, 1% Triton X-100, 10% glycerol, 25 mM NaF, 2 mM NaVO3, 5 mM Na4P2O7, protease Inhibitor cocktail (CO-RO, Roche), PH 7.4 and centrifuged. Biotin-labeled surface proteins in supernatant were precipitated by adding Streptavidin-coupled agarose beads, and dissolved in 2 X protein loading buffer (20% Glycerol, 100 mM Tris-HCl, 4% SDS, 2% β-mercaptoethanol, 0.01% bromophenol blue), and analyzed by SDS-PAGE gel.

Isolation of surface AChR and their associated proteins

Request a detailed protocol

The procedure for isolating surface AChR and associated proteins were described previously (Luo et al., 2008). Briefly, live cultured myotubes were washed with cold PBS twice, and then incubated with 300 nM biotin-α-BTX in PBS containing Mg2+/Ca2+ at 4°C for 1 hr. After washing with PBS for three times, cells were lysed in RIPA buffer. Lysates were centrifuged and supernatants were incubated with streptavidin beads for 6 hr at 4°C to precipitate biotin-labeled protein. Precipitated proteins were resolved by 2 X protein loading buffer and analyzed by SDS-PAGE gel.

Virus production

Request a detailed protocol

The procedures for generating recombinant AAV virus were as described previously (Challis et al., 2019). Briefly, full length WT or Y86F mt Rapsn was subcloned into AAV vector (AAV-CMV-GFP; Addgene Catalog, 67634). AAV vectors expressing WT Rapsn or Y86F mt Rapsn were cotransfected with helper vector (From Agilent), capsid vector (pUCmini-iCAP-PHP.B; Addgene Catalog, 103002) were cotransfected into human embryonic kidney (HEK) 293 T cells. Medium was harvested twin at 72 hr and 120 hr after transfection and viruses in the medium were precipitated by 40% PEG 8000. Cells were finally harvested and lysed in SAN digestion buffer (0.5 M NaCl, 4 mM Tris, 1 mM MgCl2). Virus was purified by using a discontinuous iodixanol gradient. The titer was determined by qPCR.

Statistical analysis

Request a detailed protocol

Data were analyzed by unpaired t-test, One-way ANOVA and Two-way ANOVA. Unless otherwise indicated, data were shown as mean ± SEM. Statistical difference was considered when p<0.05.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
    Determination of the tissue distributions and relative concentrations of the postsynaptic 43-kDa protein and the acetylcholine receptor in Torpedo
    1. WJ LaRochelle
    2. SC Froehner
    (1986)
    The Journal of Biological Chemistry 261:5270–5274.
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
    The agrin hypothesis
    1. UJ McMahan
    (1990)
    Cold Spring Harbor Symposia on Quantitative Biology 55:407–418.
    https://doi.org/10.1101/SQB.1990.055.01.041
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
  73. 73
  74. 74
  75. 75
  76. 76
  77. 77
  78. 78
  79. 79
  80. 80
  81. 81
  82. 82

Decision letter

  1. Gary L Westbrook
    Senior and Reviewing Editor; Oregon Health and Science University, United States
  2. Hans Brenner
    Reviewer; Unviersity of Basel, Switzerland
  3. Markus Ruegg
    Reviewer; Biozentrum University of Basel, Switzerland

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "A novel mechanism in agrin signaling revealed by a prevalent rapsyn mutation in congenital myasthenic syndrome" for consideration by eLife. Your article has been reviewed by Gary Westbrook as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Hans Brenner (Reviewer #1); Markus Ruegg (Reviewer #3). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript examined a knockin mouse carrying a mutation (N88K) of rapsyn, a common mutation in congenital myasthenic syndrome. The homozygous KI mice died shortly after birth with NMJ deficits. The authors implicate the agrin-LRP4-MuSK tyrosine phosphorylation of rapsyn in the pathophysiology. The authors recently reported that rapsyn has E3 ligase activity, which is essentially for its role in AChR clustering. The importance of this signaling cascade is known and many of the general observations regarding rapsyn are known, the new information in this manuscript is the link of the N88K mutation to impaired rapsyn phosphorylation at Y86. The experiments appear carefully done and described and the current results are a logical extension of the authors' prior studies and provide additional details on the role of rapsyn at the NMJ.

Essential revisions:

Although the reviewers were supportive of the manuscript, a number of essential issues will need to be addressed in a revised manuscript as listed here and explained in depth in the full comments of the reviewers below. The authors should also edit the manuscript to be careful not to overstate their conclusions in the title or text of the manuscript.

Reviewer 1 – the number of observations in each experiment should be documented.

Reviewer 2 –

1. Some of the data are overinterpreted.

2. Is rapsyn tyrosine phosphorylated at E14?

3. Self-association of N88K rapsyn.

4. Basis of variability of human phenotypes relative to the KI mouse must be explained/addressed.

5. Association with cytoskeletal proteins?

6. Figure 1 – comparison needed of WT, hets and homozygous, ephys?

7. Figure 2 – quality of staining of clusters at E14 is poor.

8. Figure 5C – explain Agrin treatment protocol.

Reviewer 3 – please address the apparent discrepancy with N88K mutations in humans.

Reviewer #1:

The neuromuscular junction has for decades been a model synapse for investigating basic mechanisms of synaptic transmission and synapse development. Motor neuron growth cones differentiate into presynaptic nerve terminals at the site of n.-m. contact, and in close register, muscle fibers form high density accumulations of receptors for the transmitter acetylcholine (AChRs). Synaptic AChR clustering is driven by agrin secreted from the motor nerve terminal and activating its effector MuSK in the muscle fiber. AChR clustering ultimately involves the anchoring of AChRs to the synaptic actin cytoskeleton through the organization of a subsynaptic apparatus, a complex of transmembrane and peripheral membrane proteins. Whereas their importance for clustering has been demonstrated genetically, the nature of their interactions, i.e. purely physical and/or involved in signaling, and their respective relevance for AChR clustering have remained largely unknown, although it would be required for full molecular understanding of NMJ development. One of the major components of the subsynaptic apparatus is rapsyn, a peripheral membrane protein linking AChRs to the synaptic actin cytoskeleton via unknown mechanisms. In the absence of rapsyn, AChRs do not cluster, and different mutations in rapsyn gene are linked to potentially life-threatening NMJ malfunctions – a subgroup of NMJ diseases called congenital myasthenic syndromes (CMS).

To dissect novel mechanisms of rapsyn's function, the authors have taken advantage of an earlier finding that one form of CMS is linked to a common mutation in rapsyn, in which asparagine 88 is mutated to lysine (N88K). Specifically, they generated rapsyn N88K mutant mice and observed impairments in NMJ development and function, with reduced synapse number, AChR density, presynaptic vesicle density and reduced synaptic folds; combined this led to cyanosis and caused death within 24 hours after birth. Reminiscent synaptic phenotypes have been reported in CMS patients carrying the N88K mutations.

In molecular and cell biological experiments on heterologous systems and in cultured muscle cells expression of N88K-mutant reduced spontaneous and agrin-induced AChR clustering without an effect on total or surface rapsyn, respectively, demonstrating reduced ability of N88K to cluster AChRs. This was not due to impaired binding of N88K to AChRs.

In previous experiments, the same group found that rapsyn possesses E3 ligase activity that is required for its ability to cluster AChRs, apparently through a process involving posttranslational neddylation of the AChRs. They now show in biochemical experiments both in HEK293 cells, in cultured muscle cells and in mouse muscle carrying the N88K mutation that indeed neddylation was strongly reduced compared to cells/tissue expressing wild type rapsyn. Evidence is then presented that agrin/MuSK phosphorylates a Tyr residue (Y86) in rapsyn immediately flanking N88 in WT, but not in N88K mutant muscle, and that phosphorylation at Y86 is required for rapsyn's E3 ligase activity and for its ability to cluster AChRs.

With activity being regulated by self-association in numerous E3 ligases, the authors then successfully tested their hypothesis that self-association of rapsyn in HEK293 cells – a condition for forming aggregates in heterologous cells – was impaired with non-phosphorylatable rapsyn Y76F mutants, as it was with rapsyn N88K mutants. They validated the physiological relevance of their hypothesis in vivo, by injecting fetal N88K mutant muscles with AAV expressing WT and N88K mutant rapsyns, respectively, which mimicked the changes in AChR clustering in the genetically modified mutant compared to control WT mice. Thus, NMJ deficits in N88K mutant muscle could be rescued by w.t. rapsyn, but not with N88K rapsyn. In contrast, forced expression of Y86F rapsyn did not rescue the synaptic phenotype in N88K mutants.

Combined, the data suggest that activated MuSK phosphorylates rapsyn at Y86 which stimulates rapsyn self-association, thus activating its E3 ligase activity which is required for AChR clustering. The paper provides not only insight into the molecular mechanism agrin-induced AChR clustering at the NMJ, but also explains, at least in part, the synaptic phenotype of rapsyn N88K patients afflicted by this form of CMS.

The data presented are well documented, except that numbers of items examined (AChR clusters, animals) should be given (see e.g. (4) below). Validations by appropriate controls are convincing.

Reviewer #2:

Essential revisions:

In the present study, the authors investigated how a prevalent mutation in a well-known congenital myasthenic syndroms (CMS) causing gene (ie Rapsyn) leads to neuromuscular junction (NMJ) formation defects in rodent. Rapsyn is a peripheral membrane protein that binds directly to the skeletal muscle acetylcholine receptors (AChR) and is essential for AChR clustering in the postsynaptic membrane. However, the mechanisms underlying Rapsyn function in anchoring postsynaptic proteins largely remain unknown. Mutations in Rapsyn cause CMS, the most common mutation occurs at N88 since about 90% of Rapsyn-related CMS patients carry N88K mutation.

The authors demonstrate by genetic approaches and functional studies that the N88 residue of Rapsyn is essential for NMJ formation in mouse and provide novel mechanistic insights for Rapsyn function in postsynaptic differentiation. Rapsyn N88K mutation strongly impairs functional differentiation of NMJ as early as embryonic day 14. Evidence is presented that the mutation alters the E3 ligase activity of Rapsyn and MuSK-induced Rapsyn tyrosine phosphorylation. The authors further identify Y86 site as one of the main residues phosphorylated by MuSK and show that it is critical for Rapsyn E3 ligase activity and AChR clustering presumably by promoting Rapsyn self-association. Finally, rescue experiments using AAV-mediated Rapsyn expression in N88K mutant muscle indicate that WT-Rapsyn, but not Y86F-Rapsyn ameliorate the NMJ morphological deficits of N88K mutant thigh muscle suggesting a critical role for Y86 in Rapsyn function during NMJ formation. This study provides important mechanistic insights of the molecular mechanisms underlying Rapsyn function in NMJ formation. Although overall the data support the conclusions, some of the results and mechanisms proposed appear over-interpreted and care should be given to the phrasing all throughout the manuscript for accuracy.

1) The authors claim that their study led to discover a pathway by which signal is transduced from MuSK to Rapsyn. However, the sequential link between MuSK activation, Rapsyn self-association, E3 ligase activity and Rapsyn tyrosine phosphorylation events in AChR clustering is not clear and no evidence is provided to support the sequential model proposed by the authors in the discussion that may only reflect one possibility among many others. The author should clarify their signaling model and propose experimental data that support their conclusions as to the mechanistic frame of Rapsyn function in AChR clustering.

2) The authors demonstrate that Rapsyn N88K mutation impairs NMJ formation in vivo as early as E14, where only minimal agrin/MuSK signaling is occurring. It would be important to determine whether Rapsyn is tyrosine phosphorylated by MuSK at this early stage and discuss how MuSK leads to Rapsyn tyrosine phosphorylation and self-association in absence of agrin.

3) N88K mutation is located in the third TPR repeat known to promote Rapsyn oligomerization. However, data previously reported that the N88K mutation does not alter the ability of Rapsyn to self-associate in heterologous cells (Ohno et al., 2002) in contrast to the authors findings. This should be included and commented in the manuscript.

4) CMS patients with Rapsyn N88K mutation are either homozygous for N88K or heteroallelic for N88K and a second mutation in the Rapsyn coding region. Strikingly, clinical reports suggest that disease severity of patients with Rapsyn compound allelic mutations is greater than that of patients with homozygous N88K Rapsyn mutation. The authors should discuss their findings based on available clinical data on the effect of N88K mutation on the variability and severity of Rapsyn-related CMS.

5) A well-known function of Rapsyn is to anchor postsynaptic proteins in the subsynaptic cytoskeleton including AChR. Interactions between Rapsyn with microtubule and actin binding proteins have been reported in the literature. It is somehow surprising that the authors did not investigate (immunostaining) whether N88K mutation impairs Rapsyn association with cytoskeleton components.

Figure 1: The authors analyzed the NMJ phenotype of mice carrying Rapsyn N88K mutation. For a meaningful comparison of the NMJ phenotypes it is important to compare WT, heterozygous and homozygous mice for the mutation. To support the conclusions of the NMJ staining and EM data, the authors could perform ex vivo electrophysiological recording at P0 to evaluate the neurotransmission defects. This may be challenging since the mutant mice died at birth, but worth to try.

Figure 2: The staining of AChR clusters at E14 is of poor quality. The authors should provide a more suitable confocal image that reflects their quantification (almost no AChR clusters are visible in the mutant).

Fig5C: The authors should explain why a 2H treatment of agrin was used to evaluate the effect of N88K mutation in Rapsyn tyrosine phosphorylation in muscle cells.

Reviewer #3:

This paper describes the effect of the most frequent mutation (N88K) in the gene coding for the adaptor molecule rapsyn when tested in mice. The authors find that mice expressing the N88K-mutated rapsyn die within 24 hours after birth because of severe perturbation of the neuromuscular junction. The authors then show that N88K-rapsyn affects neddylation, rapsyn self-association and rapsyn-phosphorylation. Interestingly, prevention of phosphorylation of the nearby tyrosine (Y86) has a very similar effect as the N88K mutant.

Overall, the paper is well done and it reports on an interesting mechanism. Having said this, I find it a bit disturbing that others have reported much milder effects of the N88K mutant (see for example Cossins et al., 2006) in similar cell assays. These contributors also reported that the N88K-mutant rapsyn affects the stability of agrin-induced AChR clusters. Finally, the N88K mutant also has a much milder phenotype in human patients. Hence, a more thorough discussion and assessment what could account for these differences would strengthen the paper. One possibility that could explain the difference in the severity might be that levels of the N88K rapsyn mutant were much lower in the current study than in those studies conducted by others. Hence, a proper dose-response comparison between mutant and wild-type rapsyn may help address this difference.

https://doi.org/10.7554/eLife.49180.031

Author response

Essential revisions:

Reviewer #1:

The neuromuscular junction has for decades been a model synapse for investigating basic mechanisms of synaptic transmission and synapse development.

[…] The data presented are well documented, except that numbers of items examined (AChR clusters, animals) should be given (see e.g. (4.) below). Validations by appropriate controls are convincing,.

We thank Dr. Brenner for his constructive comments that have significantly improved the manuscript.

Reviewer #2:

Essential revisions:

[…] The authors demonstrate by genetic approaches and functional studies that the N88 residue of Rapsyn is essential for NMJ formation in mouse and provide novel mechanistic insights for Rapsyn function in postsynaptic differentiation. Rapsyn N88K mutation strongly impairs functional differentiation of NMJ as early as embryonic day 14. Evidence is presented that the mutation alters the E3 ligase activity of Rapsyn and MuSK-induced Rapsyn tyrosine phosphorylation. The authors further identify Y86 site as one of the main residues phosphorylated by MuSK and show that it is critical for Rapsyn E3 ligase activity and AChR clustering presumably by promoting Rapsyn self-association. Finally, rescue experiments using AAV-mediated Rapsyn expression in N88K mutant muscle indicate that WT-Rapsyn, but not Y86F-Rapsyn ameliorate the NMJ morphological deficits of N88K mutant thigh muscle suggesting a critical role for Y86 in Rapsyn function during NMJ formation. This study provides important mechanistic insights of the molecular mechanisms underlying Rapsyn function in NMJ formation. Although overall the data support the conclusions, some of the results and mechanisms proposed appear over-interpreted and care should be given to the phrasing all throughout the manuscript for accuracy.

We thank the reviewer for their comments “This study provides important mechanistic insights on the molecular mechanisms underlying Rapsyn function in NMJ formation” and constructive critiques and suggestions that have significantly improved the manuscript.

1) The authors claim that their study led to discover a pathway by which signal is transduced from MuSK to Rapsyn. However, the sequential link between MuSK activation, Rapsyn self-association, E3 ligase activity and Rapsyn tyrosine phosphorylation events in AChR clustering is not clear and no evidence is provided to support the sequential model proposed by the authors in the discussion that may only reflect one possibility among many others. The author should clarify their signaling model and propose experimental data that support their conclusions as to the mechanistic frame of Rapsyn function in AChR clustering.

This is an insightful question. We have performed additional experiments to characterize the time courses of MuSK activation, Rapsyn tyrosine phosphorylation, Rapsyn self-association, Rapsyn E3 ligase activity in C2C12 myotubes in response to Agrin stimulation. As shown in revised Figure 6A, 6B and Figure 7I, 7J, MuSK activation occurred within 10 minutes, followed by Rapsyn tyrosine phosphorylation and self-association (CO-IP of Rapsyn-HA and Rapsyn-EGFP in C2C12 cells) (which plateaued around 20 and 90 minutes, respectively). E3 ligase activity was not peaked until 90 min after Agrin stimulation (Figure 6B). These data demonstrate that these events occur sequentially. Together with other data presented in this paper, they support a working model that MuSK stimulates AChR cluster formation by increasing Rapsyn phosphorylation and self-association and thus enhancing its E3 ligase activity. These results are now presented in subsection “Reduced Rapsn tyrosine phosphorylation by N88K mutation” and subsection “Y86 phosphorylation for E3 ligase activity and AChR clustering”.

2) The authors demonstrate that Rapsyn N88K mutation impairs NMJ formation in vivo as early as E14, where only minimal agrin/MuSK signaling is occurring. It would be important to determine whether Rapsyn is tyrosine phosphorylated by MuSK at this early stage and discuss how MuSK leads to Rapsyn tyrosine phosphorylation and self-association in absence of agrin.

As suggested, we looked at Rapsyn tyrosine phosphorylation in E14 muscles. As shown in Figure 6E, 6G, tyrosine-phosphorylation was detected in muscles of E14 WT mice; but the level was reduced in muscles from N88K mice. As demonstrated in the literature, both MuSK kinase activity and Rapsyn are necessary for prepatterning or the formation of aneural AChR clusters (Lin et al., 2001; Yang et al., 2001b); and Rapsyn is known to interact with MuSK in the absence of Agrin (Apel et al., 1997). Further, LRP4 could interact with MuSK and thus maintain its activity in the absence of Agrin (Kim et al., 2008; Zhang et al., 2008). Our data are in agreement with these observations. Together, they suggest that tyrosine phosphorylation of Rapsyn could occur in the absence of Agrin and contributes to the formation of aneural AChR clusters or muscle prepatterning. These points are now described in the revised subsection “Reduced Rapsn tyrosine phosphorylation by N88K mutation” and Discussion section.

3) N88K mutation is located in the third TPR repeat known to promote Rapsyn oligomerization. However, data previously reported that the N88K mutation does not alter the ability of Rapsyn to self-associate in heterologous cells (Ohno et al., 2002) in contrast to the authors findings. This should be included and commented in the manuscript.

Good point. However, we would like to point out that in the paper by Ohno and colleagues (2002), “self-association” was defined as a morphological event (i.e., aggregate formation). In fact, as shown in Figure 4A, N88K Rapsyn could form aggregates in cultured cells, in agreement with the previous work. However, the N88K mutation reduced the co-precipitation of Rapsyn-HA and Rapsyn-GFP, suggesting that N88 is critical to self-association (Figure 7K-N). We revised subsection “Impaired ability of N88K Rapsn in AChR clustering” to indicate that our results were in agreement with the previous report in terms of “aggregates” formation.

4) CMS patients with Rapsyn N88K mutation are either homozygous for N88K or heteroallelic for N88K and a second mutation in the Rapsyn coding region. Strikingly, clinical reports suggest that disease severity of patients with Rapsyn compound allelic mutations is greater than that of patients with homozygous N88K Rapsyn mutation. The authors should discuss their findings based on available clinical data on the effect of N88K mutation on the variability and severity of Rapsyn-related CMS.

Good point. This point is now discussed in revised Discussion section.

CMS patients carrying the N88K mutation show variable phenotypes, from serious, early-onset symptoms to mild, late-onset symptoms (Cossins et al., 2006; Milone et al., 2009; Muller et al., 2003), even in patients with homozygous N88K mutation. In severe cases, N88K heteroallelic with c.966 + 1GT, L14P, or a frameshift mutation may cause postnatal death (Maselli et al., 2003; Milone et al., 2009; Richard et al., 2003), indicating N88K mutation could have seriously detrimental effects on NMJ formation or maintenance in human subjects. Overall, the symptoms of CMS patients with homozygous N88K mutation are milder than that of patients carrying heteroallelic N88K mutation with a second mutation. These may be due to that second mutations may exert more harmful effects than that of N88K mutation. Actually, among these second mutations, coding sequence deletions or insertions are often observed, such as c.1177-1178del AA, p.V50-S55del, p.K373del, c.553-554insGTTCT (Milone et al., 2009), which may cause frameshift of coding sequence and totally disrupt Rapsn’s function.

We also note that phenotypes of N88K mt mice seem to be more severe than symptoms of CMS patients. Almost all N88K mt mice died within 24 hours of birth. A simple explanation of these observations may be genetic difference. Gene mutations identified in patients with various diseases such as Lesch-Nyhan syndrome, Lowe syndrome, galactosemia diseases often failed to reproduce clinic symptoms in mouse models (Elsea and Lucas, 2002). On the other hand, a knock-in mutation of AQP2, a gene implicated in hereditary non-X-linked nephrogenic diabetes insipidus, causes more severe phenotypes in mice than in human subjects (Yang et al., 2001a). Even in same species, effects of a mutation could have variable impacts on different strains of mice, for example, the Agrin mutation, Agrinnm380 (Bogdanik and Burgess, 2011). Furthermore, it is possible that some patients with the N88K mutation are unable to survive and thus be examined by physicians.

5) A well-known function of Rapsyn is to anchor postsynaptic proteins in the subsynaptic cytoskeleton including AChR. Interactions between Rapsyn with microtubule and actin binding proteins have been reported in the literature. It is somehow surprising that the authors did not investigate (immunostaining) whether N88K mutation impairs Rapsyn association with cytoskeleton components.

Good question. To this end, we used two methods – Rapsyn-Actin association

(Walker et al., 1984) and Triton extraction (Moransard et al., 2003). First, as shown in Figure 5B, the amount of Actin and Rapsyn associated with surface AChR was similar between N88K and WT myotubes, suggesting the mutation has little effect on actin association. Second, actin-anchored Rapsyn-AChR complexes are resistant to low concentration of detergents (Moransard et al., 2003). We found that the amount of Rapsyn that could be solubilized by Triton X-100 was similar between N88K and WT (Figure 5—figure supplement 1C and D). These results suggest that the N88K mutation may not impair Rapsyn association with Actin cytoskeleton components. These points are now described in subsection “Reduced E3 ligase activity of N88K mt Rapsn”.

Figure 1: The authors analyzed the NMJ phenotype of mice carrying Rapsyn N88K mutation. For a meaningful comparison of the NMJ phenotypes it is important to compare WT, heterozygous and homozygous mice for the mutation. To support the conclusions of the NMJ staining and EM data, the authors could perform ex vivo electrophysiological recording at P0 to evaluate the neurotransmission defects. This may be challenging since the mutant mice died at birth, but worth to try.

As suggested, we have performed electrophysiological recordings to analyze synaptic transmission in four genotypes (WT, heterozygous N88K/+, homozygous N88K and N88K/-). As shown in Figure 3 and in subsection “Aberrant NMJ formation in N88K mt mice”, there was no difference in resting membrane potentials among the genotypes; however, fibers with mEPP, the amplitude and frequency of mEPPs were reduced in N88K mt and N88K/- mt, compared with WT or N88K/+. These results support the conclusions of morphological studies.

Figure 2: The staining of AChR clusters at E14 is of poor quality. The authors should provide a more suitable confocal image that reflects their quantification (almost no AChR clusters are visible in the mutant).

As suggested, better representative images are provided in revised Figure 2I-L.

Figure 5C: The authors should explain why a 2H treatment of agrin was used to evaluate the effect of N88K mutation in Rapsyn tyrosine phosphorylation in muscle cells.

Agrin activates MuSK within 10 minutes of stimulation and tyrosine phosphorylation of rapsyn that plateaus around 20 minutes (Figure 6A). Agrin induces the formation of AChR clusters beginning around 2 hours of stimulation (Ngo et al., 2012). Therefore, we studied the effect of N88K on Rapsyn tyrosine phosphorylation at 2 hours of agrin stimulation. We revised Materials and methods section to explain 2 hours treatment of Agrin for evaluating tyrosine phosphorylation of Rapsyn (subsection “C2C12 culture and Generation of Gene-modified C2C12 cells”).

Reviewer #3:

[…] Overall, the paper is well done and it reports on an interesting mechanism. Having said this, I find it a bit disturbing that others have reported much milder effects of the N88K mutant (see for example Cossins et al., 2006) in similar cell assays. These contributors also reported that the N88K-mutant rapsyn affects the stability of agrin-induced AChR clusters. Finally, the N88K mutant also has a much milder phenotype in human patients. Hence, a more thorough discussion and assessment what could account for these differences would strengthen the paper. One possibility that could explain the difference in the severity might be that levels of the N88K rapsyn mutant were much lower in the current study than in those studies conducted by others. Hence, a proper dose-response comparison between mutant and wild-type rapsyn may help address this difference.

We thank Dr. Ruegg for his constructive comments that have significantly improved the manuscript.

Regarding “much milder effects of the N88K mutant (see for example Cossins et al., 2006) in similar cell assays” – These points are now discussed in the revised Discussion section –. Pathological mechanisms of the N88K mutation have been examined previously. It was shown to reduce Rapsn’s ability to induce AChR clusters in HEK and TE671 cells (Cossins et al., 2006; Ohno et al., 2002). In agreement, the N88K mutant was shown here to reduce Rapsn’s ability to induce AChR clusters in HEK293T cells (Figure 4A). In N88K knock-in mt mice and in CRISPR/Cas9-generated C2C12 myotubes, AChR clusters were almost diminished.

However, N88K seemed to be able to induce AChR clusters in Rapsn mt myotubes (Cossins et al., 2006). The cause of this discrepancy is unclear. In the current study, the impact of the N88K mutation was inferred mainly by loss-of-function approaches, both in vitro and in vivo, without changing endogenous Rapsn level (Figure 1 and Figure 4E and F). Rapsn is known to induce AChR clusters at low concentrations, but inhibit AChR clusters at high level (Han et al., 1999; Yoshihara and Hall, 1993). This inverted U-function could complicate the interpretation of results of overexpressing WT or mt Rapsn. These points are now discussed in the revised Discussion section.

Regarding “much milder phenotypes in human patients” –These points are now discussed in the revised Discussion section –. CMS patients carrying the N88K mutation show variable phenotypes, from serious, early-onset symptoms to mild, late-onset symptoms (Cossins et al., 2006; Milone et al., 2009; Muller et al., 2003), even in patients with homozygous N88K mutation. In severe cases, N88K heteroallelic with c.966 + 1GT, L14P, or a frameshift mutation may cause postnatal death (Maselli et al., 2003; Milone et al., 2009; Richard et al., 2003), indicating N88K mutation could have seriously detrimental effects on NMJ formation or maintenance in human subjects. Overall, the symptoms of CMS patients with homozygous N88K mutation are milder than that of patients carrying heteroallelic N88K mutation with a second mutation. These may be due to that second mutations may exert more harmful effects than that of N88K mutation. Actually, among these second mutations, coding sequence deletion or insertion are often observed, such as

c.1177-1178del AA, p.V50-S55del, p.K373del, c.553-554insGTTCT (Milone et al., 2009), which may cause frameshift of coding sequence and totally disrupt Rapsn’s function. We also note that phenotypes of N88K mt mice seem to be more severe than symptoms of CMS patients. Almost all N88K mt mice died within 24 hours of birth. A simple explanation of these observations may be genetic difference. Gene mutations identified in patients with various diseases such as Lesch-Nyhan syndrome, Lowe syndrome, galactosemia diseases often failed to reproduce clinic symptoms in mouse models (Elsea and Lucas, 2002). On the other hand, a knock-in mutation of AQP2, a gene implicated in hereditary non-X-linked nephrogenic diabetes insipidus, causes more severe phenotypes in mice than in human subjects (Yang et al., 2001a). Even in same species, effects of a mutation could have variable impacts on different strains of mice, for example, the Agrin mutation, Agrinnm380 (Bogdanik and Burgess, 2011). Furthermore, it is possible that some patients with the N88K mutation are unable to survive and thus be examined by physicians.

https://doi.org/10.7554/eLife.49180.032

Article and author information

Author details

  1. Guanglin Xing

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Hongyang Jing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8258-0293
  2. Hongyang Jing

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Validation, Methodology
    Contributed equally with
    Guanglin Xing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2811-1266
  3. Lei Zhang

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Data curation, Software, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  4. Yu Cao

    Department of Neuroscience and Regenerative Medicine, Augusta University, Augusta, United States
    Contribution
    Data curation, Software, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3708-575X
  5. Lei Li

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
  6. Kai Zhao

    1. Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    2. Department of Neuroscience and Regenerative Medicine, Augusta University, Augusta, United States
    Contribution
    Data curation, Software, Methodology
    Competing interests
    No competing interests declared
  7. Zhaoqi Dong

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Data curation, Software
    Competing interests
    No competing interests declared
  8. Wenbing Chen

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Data curation, Software
    Competing interests
    No competing interests declared
  9. Hongsheng Wang

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  10. Rangjuan Cao

    Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  11. Wen-Cheng Xiong

    1. Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    2. Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, United States
    Contribution
    Conceptualization, Project administration
    Competing interests
    No competing interests declared
  12. Lin Mei

    1. Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, United States
    2. Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, United States
    Contribution
    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    lin.mei@case.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5772-1229

Funding

National Institutes of Health (NS082007)

  • Lin Mei

National Institutes of Health (NS090083)

  • Lin Mei

National Institutes of Health (AG051510)

  • Lin Mei

Veterans Health Administration office of Research and Development (1/01IB001020A)

  • Lin Mei

National Institutes of Health (AG45781)

  • Wen-Cheng Xiong

National Institutes of Health (AG060997)

  • Wen-Cheng Xiong

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Peter Noakes (The University of Queensland, School of Biomedical Science) for providing Rapsn null mutants; Dr. Lin Gan (Rochester Mouse Genome Editing Resource Core) for generating N88K mt mice; members of Mei/Xiong Lab for critical comments. This study was supported in part by grants from NIH and Veterans Affairs to LM and WCX.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animals were handled according to the approved institutional animal care and use committee (IACUC) protocol (2017-0115) of Case Western Reserve University.

Senior and Reviewing Editor

  1. Gary L Westbrook, Oregon Health and Science University, United States

Reviewers

  1. Hans Brenner, Unviersity of Basel, Switzerland
  2. Markus Ruegg, Biozentrum University of Basel, Switzerland

Publication history

  1. Received: June 9, 2019
  2. Accepted: September 23, 2019
  3. Accepted Manuscript published: September 24, 2019 (version 1)
  4. Version of Record published: October 7, 2019 (version 2)

Copyright

© 2019, Xing et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,025
    Page views
  • 248
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Neuroscience
    Neven Borak, Johannes Kohl
    Insight

    Unexplained repeated pregnancy loss is associated with an altered perception of male odors and differences in brain regions that process smells.

    1. Neuroscience
    Liron Rozenkrantz et al.
    Research Article

    Mammalian olfaction and reproduction are tightly linked, a link less explored in humans. Here, we asked whether human unexplained repeated pregnancy loss (uRPL) is associated with altered olfaction, and particularly altered olfactory responses to body-odor. We found that whereas most women with uRPL could identify the body-odor of their spouse, most control women could not. Moreover, women with uRPL rated the perceptual attributes of men's body-odor differently from controls. These pronounced differences were accompanied by an only modest albeit significant advantage in ordinary, non-body-odor-related olfaction in uRPL. Next, using structural and functional brain imaging, we found that in comparison to controls, most women with uRPL had smaller olfactory bulbs, yet increased hypothalamic response in association with men's body-odor. These findings combine to suggest altered olfactory perceptual and brain responses in women experiencing uRPL, particularly in relation to men's body-odor. Whether this link has any causal aspects to it remains to be explored.