Preserving neuromuscular synapses in ALS by stimulating MuSK with a therapeutic agonist antibody
- NYU Medical School, United States
- Columbia University, United States
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 …
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) …
Figure 2
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
Figure 6
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 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (mouse) | Mouse: B6.Cg-Tg(SOD1*G93A)1Gur/J | The Jackson Laboratory | RRID:IMSR_JAX:004435 | |
| strain, strain background (mouse) | Mouse: C57BL/6J | The Jackson Laboratory | RRID:IMSR_JAX000664 | |
| cell line (mouse) | Mouse C2C12 skeletal muscle cells | Burden lab | ATCC Cat# CRL-1772, RRID:CVCL_0188 | |
| cell line (mouse) | Mouse: Immortalized wild type muscle cells | Burden lab | PMID: 18848351 | |
| cell line (mouse) | Mouse: Immortalized Lrp4 mutant muscle cells | Burden lab | PMID: 18848351 | |
| antibody | Rabbit anti-Neurofilament-L | SYnaptic SYstems | Cat# 171 002, RRID:AB_887743 | |
| antibody | Rabbit anti-Synapsin 1/2 | SYnaptic SYstems | Cat# 106 002, RRID:AB_887804 | |
| antibody | Alexa 647-anti-human IgG | Life Technologies | Cat# A-21445, RRID: AB_2535862 | |
| antibody | anti-Choline Acetyltransferase | Millipore | Cat# AB144P-200UL, RRID:AB_90661 | |
| antibody | anti-NeuN | Millipore | Cat# MAB377, RRID: AB_2298772 | |
| antibody | Rabbit anti-MuSK | Burden lab | PMID: 10781064 | |
| antibody | Goat anti-MuSK | R and D Systems | Cat# AF3904, RRID:AB_2147242 | |
| antibody | Rabbit anti-Dok-7 | Burden lab | PMID: 18848351 | |
| antibody | Mouse anti-phosphotyrosine 4G10 | Millipore | ||
| peptide, recombinant protein | Alexa 594-alpha-bungarotoxin | Life Technologies | Cat#B13423 | |
| peptide, recombinant protein | Alexa 488-alpha-bungarotoxin | Life Technologies | Cat#B13422 | |
| peptide, recombinant protein | Hoechst 33342 | Thermo Fisher | Cat# 62249 | |
| peptide, recombinant protein | Recombinant Rat Agrin Protein | R and D Systems | Cat#550-AG-100 | |
| commercial assay or kit | GAPDH TaqMan Assay Mm00186822_cn | Thermo Fisher | Cat# 4400291 | |
| software, algorithm | Prism 7.0 | http://www.graphpad.com/scientific-software/prism/ | RRID:SCR_002798 | |
| software, algorithm | Volocity 3D Image Analysis Software | http://www.perkinelmer.com/pages/020/cellularimaging/products/volocity.xhtml | RRID:SCR_002668 | |
| software, algorithm | pCLAMP Software Suite | https://www.moleculardevices.com/systems/axon-conventional-patch-clamp/pclamp-11-software-suite | ||
| other | DietGel 76A | ClearH20 | Cat#72-07-5022 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.34375.015
