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 for-mation, 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 patho-physiological 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 hLRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood.
Myasthenia gravis (MG) is the most common neuromuscular junction (NMJ) disorder, affecting 20 per 100,000 people in various populations (1-3). MG patients show characteristic fatiguing weakness of voluntary muscles, including ocular, bulbar, and limb muscles; weight loss from dysphagia; and, in severe cases, death from breathing difficulty. In a majority of patients, MG appears to stem from an autoimmune response against acetylcholine receptors (AChRs), which are critical for neurotransmission at the NMJ. Autoantibodies against AChRs can be detected in 80%-85% of MG patients (4, 5). Evidence from classic experiments indicates the anti-AChR antibodies are pathogenic (6-15). In rabbit, mouse, and rat models of experimental autoimmune MG (EAMG), anti-AChR antibodies block AChR activity (8, 11) and may acceler-ate AChR internalization and degradation (7). AChR deficiency decreases amplitudes of endplate potentials (EPPs) and miniature EPPs (mEPPs), consequently reducing the safety margin of neuromuscular transmission (9, 11). The autoantibodies may fix complements and attract macrophages, which could mediate NMJ destruction (5, 10, 16-18).
However, AChR antibodies are not detectable in approximately 20% of MG patients. Evidence indicates that these "seronegative" MG patients may generate autoantibodies against proteins critical for NMJ formation or maintenance. Agrin released from motor neurons binds to low-density lipoprotein receptor-related protein 4 (LRP4) and activates the receptor tyrosine kinase MuSK to direct NMJ formation, including AChR concentration, in the postjunctional membrane (19-27). Approximately 40%-50% of seronegative patients have antibodies against MuSK (28-30). Immunization with the extracellular domain of MuSK causes MG in rodents and rabbits (31-36). Passive transfer of IgG from anti-MuSK-positive MG patients causes MG in adult animals (37-41). The remaining 6%-12% of MG patients are double-seronegative for anti-AChR and anti-MuSK antibodies.
LRP4, a member of the low-density lipoprotein receptor (LDLR) family, contains an enormously large extracellular N-terminal region that possesses multiple EGF repeats and LDLR repeats, a transmembrane...