LRP4 is critical for neuromuscular junction maintenance

Abstract

The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.

Keywords: AChRs; LRP4; NMJ; agrin; congenital myasthenic syndrome; synaptic basal lamina.

Figure 1.

LRP4 is depleted from NMJs following conditional deletion in adults. A, Genotypes and LRP4 deletion in skeletal muscle. In ACTA1-rtTA; tetO-cre transgenic mice (Rao and Monks, 2009), cre is expressed under control of the ACTA1 (human actin α1, skeletal muscle) promoter, whereas cre expression is controlled by tetracycline inducible element (tetO). The bitransgenic mice do not express Cre until doxycycline is administered. ACTA1-rtTA; tetO-cre mice were bred with LRP4^f/f mice (Wu et al., 2012b) to generate ACTA1-rtTA; tetO-cre; LRP4^f/f (imKO). Dox-treated imKO mice did not express LRP4. Unless otherwise specified, LRP4^f/f mice were used as control. B, Reduction of LRP4 in Dox-treated imKO mice. Tibialis anterior muscle was isolated from mice of indicated genotypes and treatment and blotted for LRP4, MuSK, and β-actin. C, Dox-treated imKO mice progressively lost weight, compared with imKO or LRP4^f/f+DOX mice; n = 3 per genotype (two-way ANOVA). *p < 0.05 (Student's t test). D, Decreased grip strength in Dox-treated imKO mice; n = 3 per genotype (two-way ANOVA). *p < 0.05 (Student's t test). E, Dox-treated imKO mice were considerably smaller in size, compared with imKO mice, and developed scoliosis after 30 d of Dox treatment. F, Kaplan–Meier survival curves of control and Dox-treated imKO mice. χ² = 22.67, p < 0.0001 (Log-Rank; Mantel–Cox test).

Figure 2.

Fragmented NMJs with reduced AChR in Dox-treated imKO mice. A, TA muscles were stained whole mount with R-BTX to label AChR (red). In Dox-treated imKO mice, the endplates were smaller in size, fragmented, and displayed reduced R-BTX fluorescent intensity, compared with controls. Scale bar, 10 μm. B, Quantitative analysis revealed more fragmented AChR clusters, AChR-rich endplate area per synapse (area/NMJ), area per fragment, and AChR intensity in clusters (measured by mean pixel value). **p < 0.01 (Student's t test). n = 10. ns, Not significant. Scale bar, 10 μm.

Figure 3.

CMAP reduction in Dox-treated imKO mice. CMAPs were recorded in gastrocnemius in response to a train of 10 submaximal stimuli at different frequencies. The first stimulus response in control mice was assigned as 100%. A, Representative CMAP traces in response to the first, second, and 10th stimuli. B, All 10 CMAP traces, shown stacked in succession for better comparison. C, Reduced CMAP amplitudes at 40 Hz. D, CMAP amplitudes of the 10th stimulation at different stimulation frequencies. n = 4 mice per group. *p < 0.05 (Student's t test).

Figure 4.

Impaired neuromuscular transmission in Dox-treated imKO mice. A, Representative mEPP traces. Underlined regions in the top were enlarged in the bottom. B, C, Reduced mEPP amplitudes (B) and frequencies (C); n = 6 in each group. D, Reduced EPP amplitude and representative traces; n = 5 in each group. E, F, Increased EPP rising time (E) and half-width (F); n = 5 NMJs in each group. G, Increased pair-pulse facilitation; n = 7 NMJs in each group. *p < 0.05 (Student's t test). **p < 0.01 (Student's t test).

Figure 5.

Abnormal NMJ ultrastructure in Dox-treated imKO mice. A, Representative EM NMJ images of control and Dox-treated imKO mice. NMJs in control mice displayed typical NMJ structures, where the nerve terminals were covered by terminal Schwann cells (SC) and enriched with synaptic vesicles (SV). Occasionally, fused SVs were present at the active zones (inset, arrows). Postsynaptic membranes invaginated to form junctional folds (JF), whose crests were electron dense and contained AChR-rich regions (arrows). In Dox-treated imKO mice, the numbers of SVs, JFs (asterisks), and active zones were reduced. B, Quantification of various synaptic attributes (n = 18, for each genotype), *p < 0.05 (Student's t test). **p < 0.01 (Student's t test). ns, Not significant. Scale bar, 500 nm.

Figure 6.

Partial denervation and extensive sprouting at Dox-treated imKO NMJs. In control NMJs, AChR clusters were completely innervated by nerve terminals labeled by neurofilament (NF) and synaptophysin (syn) (green). The NMJs were covered almost entirely by terminal Schwann cells and their processes (labeled by S100β in cyan). None or negligible sprouting was observed for nerve terminals and Schwann cell processes (A, B). In Dox-treated imKO mice, NMJs became fragmented and lost innervation (pink arrow); yellow represents lost innervation and AChR clusters. Stars indicate sprouting of nerve terminals and Schwann cells; n = 30 for each genotype. **p < 0.01 (Student's t test). Scale bar, 10 μm.

Figure 7.

Loss of agrin and synaptophysin from Dox-treated imKO NMJs. A–G, Muscles were stained whole mount with antibodies against syn, α-dystroglycan or agrin, or with Alexa488-conjugated Fasciculin II. Muscle sections were stained with antibodies against MuSK, rapsyn, or utrophin. Dox-treated imKO NMJs showed reduced syn or agrin, but no change in Fasciculin II, α-dystroglycan, MuSK, rapsyn, or utrophin. H, Quantification of fluorescent intensity; n = 15. *p < 0.05 (Student's t test). **p < 0.01 (Student's t test). I, R-BTX area covered by individual synaptic proteins in in Dox-treated imKO NMJs; n = 15. *p < 0.05 (Student's t test). **p < 0.01 (Student's t test). ns., Not significant. Scale bar, 10 μm.

Figure 8.

Time course depletion of synaptic agrin and synaptophysin from the NMJs in Dox-treated imKO mice. Whole-mount staining was performed as in Figure 7. A–F, Confocal images of NMJs at indicated times. A gradual reduction of agrin and syn was observed in Dox-treated imKO NMJs. Fasciculin II levels remained unchanged. G, Quantitative analysis of loss of agrin and Syn, relative to R-BTX (n = 6). H, Quantitative analysis of loss of Agrin and Syn, relative to Fasciculin II (n = 6). Scale bar, 10 μm.

Figure 9.

Loss of the 95 kDa agrin fragment in Dox-treated imKO muscle. A, Time course of alteration of various synaptic proteins. Muscle homogenates of indicated mice were subjected to Western blotting with respective antibodies. B, Quantitative analysis of data in A.