Autoantibodies to agrin in myasthenia gravis patients

Abstract

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ.

Figure 1. Detection of agrin autoantibodies in MG patient samples.

Optical density readings of normal human serum were 0.18 ± 0.16 (mean ± SD, n  =  25). The green dotted line was set as mean + 3 SD to indicate the cut-off. The red dots indicate positive for agrin antibodies. NHS, normal human serum; OND, other neurological diseases, n  =  55; MG, myasthenia gravis, n  =  93.

Figure 2. Distribution of agrin autoantibodies among MG patients.

Of 93 MG samples previously analyzed for antibody to AChR, MuSK and LRP4, 83 were AChR+/MuSK-/LRP4-; 4 were triple seronegative (AChR-/MuSK-/LRP4-) and 6 were AChR-/MuSK+/LRP4-. The cut-off, indicated by the green line, was set as mean + 3 SD. The red dots indicate positive for agrin antibodies.

Figure 3. Recognition of agrin protein by serum samples with agrin autoantibodies.

Conditioned media from Flag-agrin-transfected HEK293 cells were incubated with serum samples with agrin antibodies or normal human serum samples. Immunocomplex and conditioned media (to indicate equal amounts of input) were subjected to Western blotting with anti-Flag antibody.

Figure 4. Recognition of agrin from transfected HEK293 cells by agrin+ serum.

Agrin positive serum 2–17 stained positively with HEK293 cells transfected with Flag-agrin construct, co-staining with anti-Flag antibody. Normal human serum cannot recognize agrin-transfected cells.

Figure 5. Serum samples with agrin antibodies inhibit agrin-induced MuSK phosphorylation and AChR clustering in myotubes.

A, Anti-agrin autoantibodies inhibit agrin-induced MuSK phosphorylation. C2C12 myotubes were incubated without or with agrin and serum samples. Endogenous MuSK was precipitated by MuSK antibody and its phosphorylation was examined by 4G10 antibody. NS, no serum. B, Anti-agrin autoantibodies inhibit agrin-induced AChR clustering. Representative images. C, Quantitative data of basal (W/O agrin, green) and induced (W/agrin, red) AChR clusters. Data shown were mean ± SEM. *, p < 0.05, compared with control.