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 devastating neurodegenerative disorder characterized by the progressive loss of upper and lower motor neurons. Despite extensive research, the pathogenesis of ALS remains unclear, and there is no effective treatment available. This lack of understanding underscores the urgent need for potential biomarkers that can help classify the disease and identify new therapeutic targets. One promising avenue of research involves the investigation of autoantibodies, particularly those targeting the low-density lipoprotein receptor-related protein 4 (LRP4). Previous studies have shown that anti-LRP4 antibodies are present in a subset of ALS patients, suggesting a possible role in the disease’s pathophysiology. This study aims to explore the presence of anti-LRP4 antibodies in a Chinese cohort of ALS patients and to investigate their correlation with neuromuscular junction (NMJ) dysfunction.
The study was conducted at Xuanwu Hospital, Capital Medical University, Beijing, China, and was approved by the institutional review board. All participants provided written informed consent. The cohort consisted of 56 ALS patients who met the El-Escorial criteria for possible, probable, or definite ALS. These patients were evaluated using the revised ALS functional rating scale (ALSFRS-r) and the disease progression rate, calculated as (48 – ALSFRS-r)/duration (months). A control group was also included, comprising 65 patients with myasthenia gravis (MG), 60 patients with other neuroimmune diseases, and 63 healthy volunteers.
Anti-LRP4 antibodies were detected using a cell-based assay. Briefly, HEK-293T cells were transfected with human CMV6-LRP4-tGFP or pEGFP as a control, using Lipofectamine 2000. The cells were cultured for 48 hours at 37°C in 5% CO2, fixed with ice-cold methanol, and blocked with 5% goat serum for 2 hours at room temperature. The cells were then incubated overnight at 4°C with patients’ sera at dilutions of 1:100 or 1:200, followed by incubation with Alexa Fluor 568-conjugated anti-human immunoglobulin G (IgG) antibody for 1.5 hours at room temperature. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 10 minutes. Digital images were acquired using a confocal laser scanning microscope, and 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.
Repetitive nerve stimulation (RNS) studies were conducted to evaluate NMJ defects. 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 in amplitude by 10% or more. All ALS patients were examined by the same technician, who was blinded to the patients’ antibody status.
The results revealed that three out of 56 (5.4%) ALS patients had anti-LRP4 antibodies, with titers of 1:800, 1:800, and 1:1600, respectively. In one patient who was available for retesting six months later, anti-LRP4 antibodies had increased by 25% as her condition worsened. Among the control groups, only one patient with MG was positive for anti-LRP4 antibodies (titer of 1:800). No anti-LRP4 antibodies were found in patients with other neuroimmune diseases or in healthy subjects.
Interestingly, all three anti-LRP4-positive ALS patients showed impaired neuromuscular transmission as measured by RNS. In contrast, among the seronegative ALS patients, 27 out of 53 (50.9%) also exhibited a definite decrement in RNS response, with some showing decrements as high as 35%. This suggests that while anti-LRP4 antibodies may contribute to NMJ dysfunction, they are not the sole factor responsible for such impairments in ALS patients.
The presence of anti-LRP4 antibodies in ALS patients has been previously reported in Greek, Italian, and American cohorts, with prevalence rates of approximately 23% and 10%, respectively. The lower prevalence observed in this Chinese cohort (5.4%) may reflect ethnic differences or variations in clinical data, such as the mean disease duration, which was 16 months in this study compared to 36 months in the Greek and Italian cohorts.
LRP4 is a transmembrane protein of the low-density lipoprotein receptor family, predominantly localized at the post-synaptic membrane of NMJs. It plays a crucial role in the clustering of acetylcholine receptors and the formation of NMJs. Anti-LRP4 antibodies have been shown to cause abnormal structures and dysfunction of the NMJ in animal studies. The findings of this study suggest that anti-LRP4 antibodies may contribute to NMJ dysfunction in ALS patients, although other factors are likely involved as well.
In conclusion, this study reports the presence of anti-LRP4 antibodies in a subset of Chinese ALS patients and highlights their potential role in NMJ dysfunction. The lower prevalence of these antibodies in the Chinese cohort compared to other populations may indicate ethnic differences or variations in clinical characteristics. Further research is needed to elucidate the precise mechanisms by which anti-LRP4 antibodies contribute to ALS pathogenesis and to explore their potential as biomarkers or therapeutic targets.
doi.org/10.1097/CM9.0000000000000284
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