Presence of Antibodies Against Low-Density Lipoprotein Receptor-Related Protein 4 and Impairment of Neuromuscular Junction in a Chinese Cohort of Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a complex and devastating disorder. It involves the loss of both upper and lower motor neurons, and currently, there is no clear understanding of its pathogenesis, nor are there effective treatments. This makes the search for potential biomarkers extremely important. These biomarkers could help classify the disease and potentially lead to the discovery of new drug targets.
In previous research, auto-antibodies against the low-density lipoprotein receptor-related protein 4 (LRP4), known as anti-LRP4 antibodies, have been found in different populations. For instance, in Greek and Italian ALS cohorts, approximately 23% of patients had these antibodies [1]. In the American ALS population, the rate was 10% [2]. Anti-LRP4 antibodies were also previously identified in myasthenia gravis (MG), which is the most common neuromuscular junction (NMJ) disorder. Animal studies have shown that these antibodies can cause NMJ abnormalities [3]. Given this background, a group of researchers from Xuanwu Hospital, Capital Medical University, Beijing, China, along with colleagues from the Mayo Clinic, Rochester, MN, USA, decided to study anti-LRP4 antibodies in Chinese patients with ALS and investigate the correlation between these antibodies and abnormal neuromuscular transmission in ALS.
The study was approved by the institutional review board of Xuanwu Hospital. All participants gave written informed consent and were enrolled between May 2015 and December 2016. Fifty-six patients with ALS who met the El-Escorial criteria for possible, probable, or definite ALS were included. They were evaluated using the revised ALS functional rating scale score (ALSFRS-r) and the rate of disease progression (calculated as (48 – ALSFRS-r)/duration (months)).
Blood samples from these patients were tested for anti-LRP4 antibodies using a cell-based assay. The results were compared to a control group. The control group consisted of 65 patients with MG, 60 patients with other neuroimmune diseases, and 63 healthy volunteers. Repetitive nerve stimulation (RNS) study, a well-known technique for evaluating NMJ defects, was used. It quantified the results of NMJ transmission at a frequency of 3 or 5 Hz. Compound muscle action potentials (CMAPs) were recorded in specific muscles (abductor digiti minimi, trapezius, and orbicularis oculi) by stimulating the corresponding nerves (ulnar, accessory, and facial nerves) at the given frequencies. A definite decremental response of CMAP was defined as a reduction in amplitude by 10% or more. All ALS patients were examined by the same technician who was blinded to the patients’ antibody status.
The cell-based assay was carried out as follows. HEK-293T cells were transfected with human CMV6-LRP4-tGFP (as a test) or with pEGFP (as a control) using Lipofectamine 2000. They were then cultured for 48 hours at 37°C in 5% CO2. The cells were fixed with ice-cold methanol and blocked with 5% goat serum. 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. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Digital images were acquired using a confocal laser scanning microscope. The samples were classified as positive or negative based on signal intensity and co-localization by two independent observers who were blinded to the patients’ RNS results. Positive samples were retested at various dilutions (1/100 – 1/3200) with the rabbit anti-LRP4 antibody, and Alexa Fluor 568-conjugated anti-rabbit IgG antibody was used as a positive control. All samples were tested at least twice.
The results showed that three out of the 56 (5.4%) patients with ALS had anti-LRP4 antibodies, with titers of 1:800, 1:800, and 1:1600 respectively. In one patient who was retested 6 months later as her condition worsened, the anti-LRP4 antibodies increased by 25%. 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. Regarding the RNS results, while thirty patients with ALS had a decreased RNS response, all three anti-LRP4-positive patients showed a positive RNS response. Among the seronegative group, 27 patients presented with a definite decrement, some with even as much as 35%, which was higher than in the seropositive subjects.
The researchers reported that anti-LRP4 antibodies were present in 5.4% of the Chinese patients with ALS in their study. However, this rate was much lower than that reported in previous studies. Given that the number of anti-LRP4 antibodies in patients with MG is lower in China than in other countries [4], they speculated that anti-LRP4 antibodies in ALS might also occur in an ethnicity-specific manner. But they could not exclude the effect of clinical data differences. For example, the mean disease course in their population was only 16 months, while it was 36 months in the Greek and Italian cohorts [1].
LRP4 is a transmembrane protein of the low-density lipoprotein receptor family. It 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]. The current 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.
In conclusion, this study provides valuable insights into the presence of anti-LRP4 antibodies in Chinese patients with ALS and their potential role in NMJ impairment. It also highlights the need for further research to better understand the complex relationship between these antibodies and ALS, especially considering the possible ethnic differences.
References: [1] Tzartos JS, Zisimopoulou P, Rentzos M, Karandreas N, Zouvelou V, Evangelakou P, et al. LRP4 antibodies in serum and CSF from amyotrophic lateral sclerosis patients. Ann Clin Transl Neurol 2014;1:80–87. doi: 10.1002/acn3.26. [2] Rivner MH, Liu S, Quarles B, Fleenor B, Shen C, Pan J, et al. Agrin and low-density lipoprotein-related receptor protein 4 antibodies in amyotrophic lateral sclerosis patients. Muscle Nerve 2017;55:430–432. doi: 10.1002/mus.25438. [3] Shen C, Lu Y, Zhang B, Figueiredo D, Bean J, Jung J, et al. Antibodies against low-density lipoprotein receptor-related protein 4 induce myasthenia gravis. J Clin Invest 2013;123:5190–5202. doi: 10.1172/jci66039. [4] Li Y, Zhang Y, Cai G, He D, Dai Q, Xu Z, et al. Anti-LRP4 autoantibodies in Chinese patients with myasthenia gravis. Muscle Nerve 2017;56:938–942. doi: 10.1002/mus.25591. [5] Weatherbee SD, Anderson KV, Niswander LA. LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction. Development 2006;133:4993–5000. doi: 10.1242/dev.02696.
doi: 10.1097/CM9.0000000000000284
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