Antibodies against low-density lipoprotein receptor-related protein 4 induce myasthenia gravis

Abstract

Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood.

Figure 1. Immunization with LRP4 induces muscle weakness in mice.

(A) Purification of ecto-LRP4. Shown are Western blot of chromatography fractions on TALON Metal Affinity resins with anti-Flag antibody. (B) Representative control and LRP4-injected A/J mice after immunization. (C) Distribution of mice with different grades of muscle weakness after each boost. (D) Reduced body weight of LRP4-injected mice with grade 2 or 3 muscle weakness. n = 8 (control); 6 (LRP4). (E) Reduced grip strength in LRP4-injected mice with grade 3 muscle weakness after boost 3. n = 8 per group. *P < 0.05; ***P < 0.001. See complete unedited blots in the supplemental material.

Figure 2. Characterization of anti-LRP4 antibodies in LRP4-injected mice.

(A) Detection of anti-LRP4 antibodies in sera of injected mice, which were collected after each boost and subjected to ELISA. n = 10 (control); 39 (boost 1); 30 (boosts 2 and 3). P < 0.001 vs. control. (B and C) Ability of mouse anti-LRP4 antibodies to recognize LRP4. Purified ecto-LRP4 (B) and lysates of cells transfected with LRP4 or empty vector control (C) were subjected to Western blot with sera from LRP4-injected mice. α-tubulin blot indicates equal loading. (D) No apparent correlation between anti-LRP4 antibody titer and muscle weakness. The y axis shows half maximum of LRP4 antibody titer, revealed by ELISA, of serial dilutions of EAMG sera. (E) NMJ staining with anti-LRP4 antibody. Normal muscle fibers were stained with sera from control or LRP4-injected mice, and immunoreactivity was visualized by Alexa Fluor 488–conjugated goat anti-mouse antibody. AChR and nuclei were stained by R-BTX and DAPI, respectively. Scale bars: 20 μm. See complete unedited blots in the supplemental material.

Figure 3. CMAP reduction in LRP4-injected 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 designated as 100%. (A) Representative CMAP traces of control and LRP4-injected mice. Shown are traces in response to the first, second, and tenth stimuli. (B) All 10 CMAP traces, shown stacked in succession for better comparison. (C and D) Reduced CMAP amplitudes at 20 Hz (C) or 40 Hz (D). (E) CMAP amplitudes of the tenth stimulation at different stimulation frequencies. n = 8 per group. *P < 0.05; **P < 0.01.

Figure 4. Impaired neuromuscular transmission in LRP4-injected mice.

(A) Representative mEPP traces. Traces at right are enlargements of underlined regions at left. (B and C) Cumulative plots of mEPP events against amplitude (B) or interval (C). (D and E) Reduced mEPP amplitude (D) and frequency (E) in LRP4-injected mice. n = 8 per group. (F) Reduced EPP amplitude in LRP4-injected mice. n = 5 per group. (G) Increased PPF in LRP4-injected mice. n = 5 per group (3–4 muscle fibers per mouse). *P < 0.05.

Figure 5. Fragmented AChR clusters and distorted axon terminals in LRP4-injected mice.

Gastrocnemius was stained whole-mount with R-BTX (red) to label AChR and antibodies against NF and SV2 (NF/SV2; green) to label nerve branches and terminals. (A and B) Collapsed z-stack images of NMJs from control and LRP4-injected mice. Lateral and top views of the reconstructed 3D images are shown at right and bottom, respectively. Scale bars: 50 μm (A); 10 μm (B). (C–F) Quantitative analysis of data by Image J. n = 3 per group. (C) Increased fragments of AChR clusters. (D) Reduced AChR area per NMJ. (E) Decreased AChR intensity. (F) Reduced overlap area of NF/SV2 and AChR staining. **P < 0.01; ***P < 0.001.

Figure 6. Abnormal NMJ ultrastructure in LRP4-injected mice.

(A) Representative electron microscopic images of diaphragm NMJs of control and LRP4-injected mice. Boxed regions are shown at higher magnification immediately below. NT, nerve terminal; MF, muscle fiber; SC, Schwann cell; SV, synaptic vesicles; JF, junctional folds. Scale bars: 1.0 μm (top); 0.5 μm (middle); 0.2 μm (bottom). (B) Reduced synaptic folding area at NMJs in sections from LRP4-injected mice. (C) Reduced synaptic vesicle density at NMJs in sections from LRP4-injected mice. (D) No change in synaptic vesicle diameter between control and LRP4-injected sections. n = 15 (control); 21 (LRP4). *P < 0.05; **P < 0.01.

Figure 7. Inhibition of agrin signaling and AChR clustering by anti-LRP4 sera.

(A) C2C12 myotubes were stimulated for 16 hours with or without agrin in the presence of sera from control or LRP4-injected mice. AChR clusters were visualized by R-BTX staining. Scale bars: 50 μm. (B) Quantification of AChR clusters >4 μm in length from A. (C) C2C12 myotubes were pretreated with sera from control or LRP4-injected mice for 3 hours prior to incubation with agrin for 30 minutes. MuSK was isolated by immunoprecipitation with anti-MuSK antibody and probed with 4G10 to reveal phospho-MuSK. Lysates were also probed directly with antibodies against MuSK and α-tubulin as input control. (D) Quantitative analysis of data in C. (E) Reduced surface LRP4 in sera-treated C2C12 myotubes. Cells were treated with sera from control or LRP4-injected mice for 1 hour. Surface protein was labeled by biotin, isolated by avidin beads, and probed with anti–ecto-LRP4 to reveal cell surface LRP4. Lysates were also probed to reveal total LRP4. (F) Quantitative analysis of data in E (3 independent experiments). *P < 0.05; **P < 0.01. See complete unedited blots in the supplemental material.

Figure 8. Complement fixation by sera of LRP4-injected mice.

(A) Subclasses of anti-LRP4 antibodies. Sera from 4 LRP4-injected mice were subjected to antibody isotyping as described in Methods. (B) Increased antibody-mediated cytotoxicity by sera from LRP4-injected mice. C2C12 myoblasts and myotubes were treated with heat-inactivated sera from control or LRP4-injected mice and guinea pig complement for 30 minutes. LDH activity was measured as described in Methods. Values were normalized to control sera (assigned as 1). Each sample was assayed in triplicate. *P < 0.05.

Figure 9. Induction of muscle weakness and NMJ impairment by passive transfer of anti-LRP4 IgGs.

IgGs were purified from control and LRP4-immunized rabbits and injected into B6/D2 mice. (A) Representative images of mice after exercise. (B) Body weight loss in anti-LRP4 IgG–injected mice. (C) Decreased muscle strength in anti-LRP4 IgG–injected mice. (D–G) Reduced CMAPs in anti-LRP4 IgG–injected mice. (D) Representative CMAP traces. (E) CMAPs at 20 Hz. (F) CMAPs at 40 Hz. (G) Reduced CMAP amplitude reduction of the tenth stimulation at different frequencies. (H) Disrupted NMJs in anti-LRP4 IgG–injected mice. Shown are collapsed z-stack NMJ images. Scale bars: 20 μm. (I and J) Quantitative analysis of data in H. n = 3 (control); 4 (anti-LRP4). *P < 0.05; **P < 0.01.