LRP4 antibodies in serum and CSF from amyotrophic lateral sclerosis patients

Abstract

Objective: Amyotrophic lateral sclerosis (ALS) and myasthenia gravis (MG) are caused, respectively, by motor neuron degeneration and neuromuscular junction (NMJ) dysfunction. The membrane protein LRP4 is crucial in the development and function of motor neurons and NMJs and LRP4 autoantibodies have been recently detected in some MG patients. Because of the critical role in motor neuron function we searched for LRP4 antibodies in ALS patients.

Methods: We developed a cell-based assay and a radioimmunoassay and with these we studied the sera from 104 ALS patients.

Results: LRP4 autoantibodies were detected in sera from 24/104 (23.4%) ALS patients from Greece (12/51) and Italy (12/53), but only in 5/138 (3.6%) sera from patients with other neurological diseases and 0/40 sera from healthy controls. The presence of LRP4 autoantibodies in five of six tested patients was persistent for at least 10 months. Cerebrospinal fluid samples from six of seven tested LRP4 antibody-seropositive ALS patients were also positive. No autoantibodies to other MG autoantigens (AChR and MuSK) were detected in ALS patients. No differences in clinical pattern were seen between ALS patients with or without LRP4 antibodies.

Conclusions: We infer that LRP4 autoantibodies are involved in patients with neurological manifestations affecting LRP4-containing tissues and are found more frequently in ALS patients than MG patients. LRP4 antibodies may have a direct pathogenic activity in ALS by participating in the denervation process.

Figure 1

Detection of LRP4 antibodies in sera of ALS patients using a cell-based assay (CBA). Immunofluorescence study of the binding of antibodies to HEK293 cells transfected with pCMV6-LRP4-tGFP or pEGFP. Left column: GFP fluorescence; middle column: bound antibody staining; right column: merged images Rows 1 and 2: Commercial rabbit LRP4 antibody (1:750 dilution) was incubated with pEGFP-transfected cells (row 1; A–C) or pCMV6-LRP4-tGFP-transfected cells (row 2; D–F) and bound antibodies visualized using Alexa 568-conjugated anti-rabbit IgG antibodies. Rows 3–5: LRP4-GFP-transfected cells were incubated with a 1:100 dilution of serum from two ALS patients (rows 3–4; G–L) or a healthy control (row 5, M–O) and bound antibodies visualized with Alexa 568-conjugated anti-human IgG antibodies. Only the commercial antibody and patients' sera bound on expressed LRP4 as visualized with red staining (E, H, K) and the merged images (F, I, L).

Figure 2

Comparison of the anti-LRP4 titers between ALS and MG anti-LRP4 positive sera. Twenty-one out of the 24 positive ALS sera were tested and compared with 11 randomly chosen LRP4-positive MG sera. Sera were tested by the CBA at dilutions 1/100, 1/200, 1/400, 1/500, 1/1000, 1/2000 and 1/4000. Ambiguous result in a certain dilution is presented as an intermediate titer (e.g., ambiguous result at 1/2000 is presented as titer 1/1500)