Presence of antibodies against low-density lipoprotein receptor-related protein 4 and impairment of neuromuscular junction in a Chinese cohort of amyotrophic lateral sclerosis

Abstract

Supplemental Digital Content is available in the text

To the Editor: Amyotrophic lateral sclerosis (ALS) is a heterogeneous disorder characterized by a loss of upper and lower motor neurons with neither clear pathogenesis nor effective treatment. Thus, potential biomarkers are needed to classify the disease and find new drug targets. Previous studies have shown that auto-antibodies against the low-density lipoprotein receptor-related protein 4 (LRP4), called the anti-LRP4 antibodies, are found in ∼23% patients of Greek and Italian ALS cohorts,^[1] and in 10% of the American ALS population.^[2] Anti-LRP4 antibodies were previously identified in myasthenia gravis (MG), the most common neuromuscular junction (NMJ) disorder, and were shown to cause NMJ abnormality in animal studies.^[3] Here, we studied anti-LRP4 antibodies in Chinese patients and investigated the correlation between anti-LRP4 antibodies and abnormal neuromuscular transmission in ALS.

This study was approved by the institutional review board of Xuanwu Hospital, Capital Medical University, Beijing, China. All study participants gave written informed consent and were enrolled from May 2015 to December 2016. Fifty-six patients with ALS met the El-Escorial criteria for possible, probable, or definite ALS, and were evaluated by the revised ALS functional rating scale score (ALSFRS-r) and rate of disease progression (disease progression rate = (48 − ALSFRS-r)/duration (months)). Anti-LRP4 antibodies were detected with a cell-based assay in blood samples. The results were compared to a control group, which consisted of 65 patients with MG, 60 patients with other neuroimmune diseases, and 63 healthy volunteers. Repetitive nerve stimulation (RNS) study, a widely-used technique for evaluating NMJ defects, was used to quantify the results of NMJ transmission at a frequency of 3 or 5 Hz. Compound muscle action potentials (CMAPs) were recorded in the abductor digiti minimi, trapezius, and orbicularis oculi muscles with stimulation of the ulnar, accessory, and facial nerves at frequencies of 3 or 5 Hz. A definite decremental response of CMAP was defined as a reduction of the amplitude by 10% or more. All patients with ALS were examined by the same technician, who was blinded to the patients’ antibody status.

Briefly, HEK-293T cells were transfected with human CMV6-LRP4-tGFP (OriGene, Rockville, MD, USA) or with pEGFP (a gift from the Cell Therapy Center, Beijing, China) as a control, with Lipofectamine 2000 (Invitrogen, Waltham, Massachusetts, USA), and cultured for 48 h at 37°C in 5% CO₂. They were fixed with ice-cold methanol and blocked with 5% goat serum for 2 h at room temperature. After incubating overnight at 4°C with patients’ sera at dilutions of 1:100 or 1:200, they were incubated with Alexa Fluor 568-conjugated anti-human immunoglobulin G (IgG) antibody (Life Technologies, Invitrogen) for 1.5 h at room temperature. The nuclei were counterstained with 4,6-diamidino-2-phenyiindole (DAPI) (1:1000) for 10 min. After mounting the cells onto slides, digital images were acquired with a confocal laser scanning microscope (TCS SP5 II; Leica, Wetzlar, Germany). The confocal settings, such as gain and offset, were kept constant to ensure that all images were collected with the same parameters. Based on the signal intensity and co-localization, the samples were classified as positive or negative by two independent observers, who were blinded to the patients’ RNS results [Figure 1]. The positive samples were then retested at various dilutions (1/100–1/3200) with the rabbit anti-LRP4 antibody (Santa Cruz Biotechnology, Dallas, Texas, USA). Alexa Fluor 568-conjugated anti-rabbit IgG antibody (Invitrogen) was used as a positive control. All samples were tested at least twice.

Figure 1.

Detection of anti-LRP4 antibodies in patients with ALS by CBA. The immunofluorescence study of the binding of antibodies to HEK-293T cells transfected with pCMV6-LRP4-tGFP or pEGFP. Positive and negative control: Commercial rabbit LRP4 antibody (1:200 dilution) was incubated with pCMV6-LRP4-tGFP-transfected cells (Row 1; A, B, C) or pEGFP-transfected cells (Row 2; D, E, F). Patient 1 and 2: LRP4-GFP-transfected cells were incubated with a 1:100 dilution of serum from two patients with ALS (Row 3; G, H, I; Row 4; J, K, L). Only the commercial antibody and patients’ sera bound on expressed LRP4 as visualized with red staining (B, H) and the merged images (C, I). ALS: Amyotrophic lateral sclerosis; Anti-LRP4 antibodies: Antibodies against LRP4; CBA: Cell-based assay; LRP4: Low-density lipoprotein receptor-related protein 4.

Three of the 56 (5.4%) patients with ALS had anti-LRP4 antibodies, with titers of 1:800, 1:800, and 1:1600, respectively. In the one patient who was available for retesting 6 months later, anti-LRP4 antibodies had increased by 25% as her condition worsened. Among the controls, only one patient with MG was positive (titer of 1:800). No anti-LRP4 antibodies were found in patients with other neuroimmune diseases or in healthy subjects. While thirty patients with ALS had a decreased RNS response, all three seropositive patients showed a positive RNS response [Supplementary Table 1]. Among the seronegative group, 27 patients presented with a definite decrement, some with even as much as 35%, a decrement higher than in the seropositive subjects.

We report anti-LRP4 antibodies in 5.4% of the Chinese patients with ALS in our study. However, this rate was much lower than that reported in previous studies.^[1,2] Given that the number of anti-LRP4 antibodies in patients with MG is lower in China than in other countries,^[4] we speculated that anti-LRP4 antibodies in ALS might also occur in an ethnicity-specific manner. However, we could not exclude the effect of clinical data differences. A major parameter, the mean disease course, was only 16 months in our populations, whereas it was 36 months in the Greek and Italian cohorts.^[1]

LRP4, a transmembrane protein of the low-density lipoprotein receptor family, is mostly concentrated at the post-synaptic membrane of NMJs. It is involved in the clustering of acetylcholine receptors and the formation of NMJs.^[5] Anti-LRP4 antibodies can lead to abnormal structures and dysfunction of the NMJ.^[3] Our results revealed that all three anti-LRP4-positive patients had impaired neuromuscular transmission as measured electrophysiologically, while nearly half of the seronegative patients also had neuromuscular dysfunction. This indicates that anti-LRP4 antibodies may be one of the factors resulting in NMJ dysfunction.

Acknowledgements

The authors thank Zhong-Feng Liu and Jing An for experimental assistance.

Conflicts of interest

None.