Characterization of LRP4/Agrin Antibodies From a Patient With Myasthenia Gravis

Abstract

Background and objective: To determine whether human anti-LRP4/agrin antibodies are pathogenic in mice and to investigate underpinning pathogenic mechanisms.

Methods: Immunoglobulin (Ig) was purified from a patient with myasthenia gravis (MG) with anti-LRP4/agrin antibodies and transferred to mice. Mice were characterized for body weight, muscle strength, twitch and tetanic force, neuromuscular junction (NMJ) functions including compound muscle action potential (CMAP) and endplate potentials, and NMJ structure. Effects of the antibodies on agrin-elicited muscle-specific tyrosine kinase (MuSK) activation and AChR clustering were studied and the epitopes of these antibodies were identified.

Results: Patient Ig-injected mice had MG symptoms, including weight loss and muscle weakness. Decreased CMAPs, reduced twitch and tetanus force, compromised neuromuscular transmission, and NMJ fragmentation and distortion were detected in patient Ig-injected mice. Patient Ig inhibited agrin-elicited MuSK activation and AChR clustering. The patient Ig recognized the β3 domain of LRP4 and the C-terminus of agrin and reduced agrin-enhanced LRP4-MuSK interaction.

Discussion: Anti-LRP4/agrin antibodies in the patient with MG is pathogenic. It impairs the NMJ by interrupting agrin-dependent LRP4-MuSK interaction.

Figure 1. Patient Immunoglobulin (Ig) Recognized Lipoprotein Receptor-Related Protein 4 (LRP4) and Agrin and Labeled the Neuromuscular Junction (NMJ)

(A) The patient Ig recognized human LRP4-ECD-Myc protein. (B) The patient Ig recognized the endogenous Lrp4 protein in C₂C₁₂. (C) The patient Ig recognized Flag-agrin-LG123 protein (a C-terminal 110-KDa fragment). (D) Staining the NMJ with the patient Ig, but not healthy Ig. Scale bars:10 μm. IP = immunoprecipitation.

Figure 2. Weight Loss, Muscle Weakness, and Twitch and Tetanus Force Decreased in Patient Immunoglobulin (Ig)–Injected Mice

(A) Scheme of passive experimental autoimmune myasthenia gravis. (B) Decreased body weight in patient Ig-injected mice, not in healthy Ig-injected mice, compared with phosphate-buffered saline (PBS)–injected control mice. (C) Declined forelimb grip strength in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (D) Increased forelimb grip fatigue in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (E) Reduced forelimbs hanging time in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (F) Decreased running time in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (G) Reduced single twitch force by muscle stimulation in patient Ig-injected mice, not in healthy IgG-injected mice, compared with PBS-injected control mice. (H) Reduced single twitch force by sciatic nerve stimulation in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (I) Representative tetanic curves at stimulation frequency 50 and 100 Hz by muscle stimulation. (J) Reduced tetanic force by muscle stimulation at different stimulation frequencies in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (K) Representative tetanic curves at stimulation frequency 50 and 100 Hz by sciatic nerve stimulation. (L) Reduced tetanic force muscle stimulation at different stimulation frequencies in patient Ig-injected mice, not healthy Ig-injected mice, compared with PBS-injected control mice. n = 3 mice per group, **p < 0.01.

Figure 3. Decreased Compound Muscle Action Potentials (CMAPs) and Presynaptic and Postsynaptic Deficits in Patient Immunoglobulin (Ig)–Injected Mice

(A) Stacked 10 succession CMAP traces at 40 Hz. (B) CMAP traces in response to the first, second, and 10th stimuli. (C) Reduced CMAP amplitudes of the 10th stimulation at different stimulation frequencies in patient Ig-injected mice, not in healthy Ig-injected mice, compared with phosphate-buffered saline (PBS)–injected control mice. (D) Reduced CMAP amplitudes at 40 Hz. (E) Representative miniature end plate potential (mEPP) traces. (F, H) Cumulative plots of mEPP events against interval (F) or amplitude (H). (G, I) Reduced mEPP frequency (G) and amplitude (I) in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (J) Reduced end plate potential (EPP) amplitude in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (K) Decreased PPR in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. n = 3 mice per group. *p < 0.05, **p < 0.01.

Figure 4. Increased Neuromuscular Junction (NMJ) Fragment, Reduced Acetylcholine Receptor (AChR) Density, and Increased Denervation in the NMJ of Patient Immunoglobulin (Ig)–Injected Mice

(A) z-Stack images of NMJs from phosphate-buffered saline (PBS)–injected control mice, healthy Ig-injected mice, and patient Ig-injected mice. Scale bars 10 μm. (B–E) Quantitative analysis of data. (B) Increased fragments of AChR clusters in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (C) Reduced AChR area per NMJ in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (D) Decreased AChR intensity in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. (E) Reduced overlap area of neurofilament-L/synapsin-1 and AChR staining in patient Ig-injected mice, not in healthy Ig-injected mice, compared with PBS-injected control mice. n = 3 mice per group. **p < 0.01.

Figure 5. Patient Immunoglobulin (Ig) Impaired Agrin-Elicited Muscle-Specific Tyrosine Kinase (MuSK) Activity and Acetylcholine Receptor (AChR) Clustering

(A, B) IgG2 as the major isotype of anti-LRP4 and anti-Agrin antibodies in patient Ig. (C) Inhibition of MuSK activation by patient Ig. Phospho-MuSK elicited by agrin (C-terminal 22-KDa fragment) in myotubes treated without or with patient Ig or healthy Ig. (D) Quantitative analysis of data in C. (E–G) Inhibition of agrin-induced AChR clustering by patient Ig. Myotubes were treated without or with agrin LG3 in the presence or absence of patient or healthy Ig and assayed for AChR clusters. (E) Representative images. Scale bars: 50 μm. (F, G) Quantification of data in E. AChR clusters >4 μm in length were scored. n = 3, **p < 0.01.

Figure 6. Patient Immunoglobulin (Ig) Recognized β3 Domain of Low-Density Lipoprotein Receptor-Related Protein 4 (LRP4) and LG1 or LG2 Domain of Agrin

(A) Schematic diagrams of LRP4 extracellular domain (ECD) and individual domains. (B, C) Identification of β3 domain as the epitope by ELISA (B) and by coprecipitation (C). (B) Healthy or patient Ig was immobilized on wells and incubated with alkaline phosphatase (AP)–tagged LRP4 proteins, and AP activity was assayed by ELISA. n = 3, **p < 0.01. (C) Lysates of HEK293 cells expressing respective LRP4 proteins were incubated with indicated Ig or anti-Flag antibody and bound proteins were detected by Western blotting. (D) Schematic diagrams of agrin constructs. (E) Agrin-LG123 and LG23, not agrin-LG3, bound patient Ig. n = 3.

Figure 7. Patient Immunoglobulin (Ig) Inhibited Agrin-Enhanced Low-Density Lipoprotein Receptor-Related Protein 4 (LRP4)–Muscle-Specific Tyrosine Kinase (MuSK) Interaction

(A) Increased LRP4-ECD-MuSK interaction by agrin. Condition medium of HEK293 cells transfected with LRP4-ECD-Myc was incubated with Flag-MuSK-ECD IgG beads in the presence or absence of agrin LG3. LRP4-ECD-Myc in the precipitates with Flag-MuSK-ECD-IgG beads was revealed by Western blotting. (B) Quantification of data in A. (C) Inhibition of agrin-enhanced LRP4–MuSK interaction by patient Ig. (D) Quantitative analysis of data in C. (E) Little effect of patient Ig on basal LRP4–MuSK interaction. (F) Quantitative data in E. n = 3, *p < 0.05, **p < 0.01.