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
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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 …

https://doi.org/10.7554/eLife.49180.002
Figure 1—source data 1

Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 1E–J.

https://doi.org/10.7554/eLife.49180.005
Figure 1—figure supplement 1
Reduced AChR cluster size, increased AChR cluster length and extensive nerve terminal arborization in N88K mt mice.

(A) Generation of N88K mt mice by CRISPR-Cas9. Top, the Rapsn gene structure. Genomic sequence flanking the Asn 88 was shown below. Guidance RNA sequence, GCT CGA AAG CTA CCT GAA CC, was labeled by …

https://doi.org/10.7554/eLife.49180.003
Figure 1—figure supplement 1—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 1—figure supplement 1C, E and H.

https://doi.org/10.7554/eLife.49180.004
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; …

https://doi.org/10.7554/eLife.49180.006
Figure 2—source data 1

Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 2E, F, G, H, M and N.

https://doi.org/10.7554/eLife.49180.007
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 recordin…

https://doi.org/10.7554/eLife.49180.008
Figure 3—source data 1

Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figures 3A, B, D and E.

https://doi.org/10.7554/eLife.49180.009
Figure 4 with 2 supplements
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 …

https://doi.org/10.7554/eLife.49180.010
Figure 4—source data 1

Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 4B and F.

https://doi.org/10.7554/eLife.49180.015
Figure 4—figure supplement 1
Comparable protein stability between WT and N88K mt Rapsn in HEK293T cells.

(A) Comparable degradation rate between WT and N88K mt Rapsn in HEK293T cells. HEK293T cells were transfected with EGFP tagged WT or N88K mt Rapsn. After 24 hr, cells were separated into 6-well …

https://doi.org/10.7554/eLife.49180.011
Figure 4—figure supplement 1—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 4—figure supplement 1B.

https://doi.org/10.7554/eLife.49180.012
Figure 4—figure supplement 2
Comparable Rapsn mRNA level and Rapsn protein stability between WT and N88K mt myotubes.

(A) Verification of N88K mutation by genomic DNA sequencing. Genomic DNA of WT and N88K mt C2C12 was sequenced and aligned. Dashed rectangle to indicate N88K mutation. (B) Comparable Rapsn mRNA …

https://doi.org/10.7554/eLife.49180.013
Figure 4—figure supplement 2—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 4—figure supplement 2B and D.

https://doi.org/10.7554/eLife.49180.014
Figure 5 with 1 supplement
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 …

https://doi.org/10.7554/eLife.49180.016
Figure 5—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 5D, F and H.

https://doi.org/10.7554/eLife.49180.019
Figure 5—figure supplement 1
Comparable binding ability of N88K mt Rapsn with subunits of AChR and Actin.

(A, B) HEK293T cells were cotransfected with HA tagged WT or N88K mt Rapsn and Flag tagged α-AChR (A) or δ-AChR (B). After 48 hr, cells were lysed and the interaction of Rapsn with subunits of AChR …

https://doi.org/10.7554/eLife.49180.017
Figure 5—figure supplement 1—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 5—figure supplement 1D.

https://doi.org/10.7554/eLife.49180.018
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 …

https://doi.org/10.7554/eLife.49180.020
Figure 6—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 6D, G and I.

https://doi.org/10.7554/eLife.49180.021
Figure 7 with 1 supplement
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 …

https://doi.org/10.7554/eLife.49180.022
Figure 7—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 7B, D, F, H, J, L and N.

https://doi.org/10.7554/eLife.49180.025
Figure 7—figure supplement 1
Comparable Rapsn mRNA level and Rapsn protein stability between WT and Y86F mt myotubes.

(A) Generation of Y86F mt C2C12 cells by CRISPR-Cas9. Top, the Rapsn gene structure. Genomic sequence flanking the Y86 was shown below. Guidance RNA targeting sequence, CAT TGC TGC GCG CCA GGT TC, …

https://doi.org/10.7554/eLife.49180.023
Figure 7—figure supplement 1—source data 1

Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 7—figure supplement 1C and E.

https://doi.org/10.7554/eLife.49180.024
Figure 8 with 1 supplement
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 …

https://doi.org/10.7554/eLife.49180.026
Figure 8—source data 1

Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 8B, C and D.

https://doi.org/10.7554/eLife.49180.028
Figure 8—figure supplement 1
Generation of AAV vectors expressing WT or Y86F mt Rapsn and examination of AAV expression.

(A) Schematic diagram of generation of AAV vector expressing Rapsn. (B) HEK293T cells were infected with AAV-Rapsn-EGFP (AAV-WT-EGFP) or AAV-Y86F-Rapsn-EGFP (AAV-Y86F-EGFP). After 24 hr, Rapsn-EGFP …

https://doi.org/10.7554/eLife.49180.027

Tables

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/

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