Specific binding of collagen Q to the neuromuscular junction is exploited to cure congenital myasthenia and to explore bases of myasthenia gravis

Abstract

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG.

Fig. 1

Schematic diagram of anchoring of ColQ to NMJ. Twelve catalytic subunits of AChE are attached to ColQ to form ColQ-tailed AChE. Two heparan sulfate proteoglycan-binding domains of ColQ are bound to perlecan. C-terminal domain of ColQ is bound to MuSK.

Fig. 2

Motor functions and compound muscle action potentials (CMAP) after intravenous injection of AAV8-COLQ to the tail vein of Colq−/− mice. (A) Durations on the rotarod that was linearly accelerated from 0 to 40 rpm in 240 sec. Six mice were studied for each group. (B) Fatigue test using the rotarod was performed on six mice in each group. (C) Voluntary movements were quantified by a counter-equipped running wheel for 24 h on 6 mice in each group. (D) CMAP amplitudes of quadriceps muscles after 2-Hz stimulation of the femoral nerve. Treatment ameliorated CMAP decrements in 3 mice in each group (P < 0.05 by two-way ANOVA). *P < 0.05 and **P < 0.01 by Bonferroni post-hoc test. Mean and SE are indicated in each panel.

Fig. 3

(A) Daily injection of the purified recombinant human AChE/ColQ protein complex into the gluteus maximus muscles of Colq−/− mice induces expression of AChE/ColQ in the non-injected triceps muscle. Scale bar = 10 μm. (B) The size of ColQ-positive area is normalized for the size of AChR-positive area at the NMJs of non-injected triceps. (C) Signal intensities of ColQ at the NMJs of non-injected triceps. Mean and SE are indicated in (B) and (C). *P < 0.001.

Fig. 4

In vitro overlay assays. Purified recombinant AChE/ColQ was overlaid on a 10-µm quadriceps muscle section of Colq−/− mice in the presence of purified IgG of a control or patients. ColQ is stained with anti-ColQ antibody and AChR with Alexa594-labeled α-bungarotoxin. Scale bar = 50 µm.

Fig. 5

In vitro plate-binding assays. Increasing amounts of MuSK-IgG block binding of the purified recombinant AChE/ColQ to the extracellular domain of human MuSK that is coated on a 96-well plate. Mean and SE of 3 experiments are plotted. *P < 0.01 between controls and patients.

Fig. 6

Passive transfer of MuSK-IgG of Ct. 2 and Pt. 2 to C57BL/6J mice. (A and B) Quadriceps muscle sections of mice injected with IgG of Ct. 2 or Pt. 2 are stained for AChR by Alexa594-labeled α-bungarotoxin, ColQ and MuSK by immunostaining, AChE by cytochemical staining. Scale bar = 40 µm. Signal areas (C), intensities (D), and densities (intensity/area) (E) of the indicted molecules per NMJ are shown in mean and SE. (F) Densities of ColQ and MuSK are normalized for the density of AChR to estimate the number of ColQ and MuSK per AChR. Open and closed bars represent Ct. 2 and Pt. 2, respectively. *P < 0.05; ***P < 0.001; ns, not significant.