Preserving neuromuscular synapses in ALS by stimulating MuSK with a therapeutic agonist antibody

  1. Sarah Cantor
  2. Wei Zhang
  3. Nicolas Delestrée
  4. Leonor Remédio
  5. George Z Mentis
  6. Steven J Burden  Is a corresponding author
  1. NYU Medical School, United States
  2. Columbia University, United States
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
MuSK agonist antibodies activate MuSK, independent of Lrp4, by binding the Fz-like domain in MuSK.

(A) C2 myotubes were treated with neural Agrin or antibodies for the indicated times. MuSK was immunoprecipitated, and Western blots were probed for MuSK or phosphotyrosine. Neural Agrin and MuSK …

https://doi.org/10.7554/eLife.34375.003
Figure 1—figure supplement 1
MuSK agonist antibody #13 stimulates MuSK tyrosine phosphorylation in vivo.

(A) The MuSK agonist antibody (Ab #13) failed to stimulate MuSK phosphorylation in innervated (Inn) muscle, suggesting that MuSK may be maximally phosphorylated at synapses by Agrin and Lrp4. (B) …

https://doi.org/10.7554/eLife.34375.004
MuSK agonist antibody #13 engages MuSK at the synapse shortly after IP injection.

(A) Staining for the injected MuSK antibody (10 mg/kg) was evident as early as three days (iv-vi) and persisted for at least seven days after a single injection of antibody (vii-ix). A human …

https://doi.org/10.7554/eLife.34375.005
Figure 3 with 1 supplement
A single injection of the MuSK agonist antibody at P90 reduced synaptic loss for twenty days in SOD1-G93A mice.

(A) AChRs are concentrated in the postsynaptic membrane at innervated and fully denervated synapses (scale bar = 20 μm). (B, C) Denervation is evident in female and extensive in male SOD1-G93A mice …

https://doi.org/10.7554/eLife.34375.006
Figure 3—figure supplement 1
hSOD1-G93A copy number remained unchanged over generations and throughout the experiments.

(A) The copy number of the human SOD1-G93A gene was quantified by real-time PCR and normalized to GAPDH. All mice included in this study had 21–26 copies of hSOD1-G93A. The normalized hSOD1-G93A Ct …

https://doi.org/10.7554/eLife.34375.007
Figure 4 with 3 supplements
Chronic dosing with the MuSK agonist antibody prevents further denervation in SOD1-G93A mice.

(A) The reverse chimera MuSK agonist antibody #13 was injected at P90 () (B) The extent of full innervation decreases progressively from P90 to P162 in female and male SOD1-G93A injected with a …

https://doi.org/10.7554/eLife.34375.008
Figure 4—figure supplement 1
The human and reverse chimera versions of MuSK agonist antibody #13 induce acetylcholine receptor (AChR) clustering in C2C12 myotubes whereas a Fab from MuSK antibody #13 fails to stimulate AChR clustering.

(A) Human (h) and reverse chimera (rc) MuSK #13 antibodies are similarly effective in stimulating AChR clustering in C2C12 myotubes (n = 3). (B,C) C2C12 myotubes were treated with rc MuSK #13 or a …

https://doi.org/10.7554/eLife.34375.009
Figure 4—figure supplement 2
The reverse chimera MuSK agonist antibody #13 has a half-life of 11 days and chronic dosing with this antibody maintains the agonist antibody at levels that are sufficient to saturate MuSK at the synapse.

Rc MuSK antibody #13 was produced to minimize the occurrence of an immune response to a human antibody, as well as to eliminate the danger of eliciting an immune response at the neuromuscular …

https://doi.org/10.7554/eLife.34375.010
Figure 4—figure supplement 3
Chronic dosing with the MuSK agonist antibody increases the extent of nerve terminal coverage at partially innervated synapses in SOD1-G93A mice.

(A) The number (~50%) of partially innervated synapses in SOD1-G93A mice is not altered by chronic injection of reverse chimera MuSK agonist antibody #13 (). The inset shows a representative …

https://doi.org/10.7554/eLife.34375.011
Figure 5 with 1 supplement
The MuSK agonist antibody improves motor system output in the diaphragm muscle.

(A,B) 20 Hz stimulation (arrow) of the phrenic nerve from SOD1-G93A mice injected with the control antibody to GP120 led to a rapid and severe decline in the CMAP amplitude. In contrast, the CMAP …

https://doi.org/10.7554/eLife.34375.012
Figure 5—figure supplement 1
Drawing of experimental protocol to stimulate the phrenic nerve and record CMAPs in the diaphragm muscle.

The phrenic nerve was placed in a bipolar suction electrode for stimulation. CMAPs were recorded from a suction recording electrode placed in the same place (dotted circle) across the different …

https://doi.org/10.7554/eLife.34375.013
Chronic dosing with the MuSK agonist antibody increases motor neuron survival and extends the lifespan of SOD1-G93A mice.

(A) Representative images of lumbar spinal cords stained with antibodies to ChAT (scale bar = 100 μm). (B) At P138, during the peak period of motor neuron cell death, the number of spinal motor …

https://doi.org/10.7554/eLife.34375.014

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
strain, strain background (mouse)Mouse: B6.Cg-Tg(SOD1*G93A)1Gur/JThe Jackson LaboratoryRRID:IMSR_JAX:004435
strain, strain background (mouse)Mouse: C57BL/6JThe Jackson LaboratoryRRID:IMSR_JAX000664
cell line (mouse)Mouse C2C12 skeletal muscle cellsBurden labATCC Cat# CRL-1772,
RRID:CVCL_0188
cell line (mouse)Mouse: Immortalized wild type
muscle cells
Burden labPMID: 18848351
cell line (mouse)Mouse: Immortalized Lrp4 mutant
muscle cells
Burden labPMID: 18848351
antibodyRabbit anti-Neurofilament-LSYnaptic SYstemsCat# 171 002,
RRID:AB_887743
antibodyRabbit anti-Synapsin 1/2SYnaptic SYstemsCat# 106 002,
RRID:AB_887804
antibodyAlexa 647-anti-human IgGLife TechnologiesCat# A-21445,
RRID: AB_2535862
antibodyanti-Choline AcetyltransferaseMilliporeCat# AB144P-200UL,
RRID:AB_90661
antibodyanti-NeuNMilliporeCat# MAB377,
RRID: AB_2298772
antibodyRabbit anti-MuSKBurden labPMID: 10781064
antibodyGoat anti-MuSKR and D SystemsCat# AF3904,
RRID:AB_2147242
antibodyRabbit anti-Dok-7Burden labPMID: 18848351
antibodyMouse anti-phosphotyrosine 4G10Millipore
peptide, recombinant proteinAlexa 594-alpha-bungarotoxinLife TechnologiesCat#B13423
peptide, recombinant proteinAlexa 488-alpha-bungarotoxinLife TechnologiesCat#B13422
peptide, recombinant proteinHoechst 33342Thermo FisherCat# 62249
peptide, recombinant proteinRecombinant Rat Agrin ProteinR and D SystemsCat#550-AG-100
commercial assay or kitGAPDH TaqMan
Assay Mm00186822_cn
Thermo FisherCat# 4400291
software, algorithmPrism 7.0http://www.graphpad.com/scientific-software/prism/RRID:SCR_002798
software, algorithmVolocity 3D Image Analysis Softwarehttp://www.perkinelmer.com/pages/020/cellularimaging/products/volocity.xhtmlRRID:SCR_002668
software, algorithmpCLAMP Software Suitehttps://www.moleculardevices.com/systems/axon-conventional-patch-clamp/pclamp-11-software-suite
otherDietGel 76AClearH20Cat#72-07-5022

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