Serum Reactive Antibodies against the N-Methyl-D-Aspartate Receptor NR2 Subunit-Could They Act as Potential Biomarkers?

Abstract

Synaptic dysfunction and disrupted communication between neuronal and glial cells play an essential role in the underlying mechanisms of multiple sclerosis (MS). Earlier studies have revealed the importance of glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) receptor, in excitotoxicity, leading to abnormal synaptic transmission and damage of neurons. Our study aimed to determine whether antibodies to the NR2 subunit of NMDAR are detected in MS patients and evaluate the correlation between antibody presence and clinical outcome. Furthermore, our focus extended to examine a possible link between NR2 reactivity and anti-coagulant antibody levels as pro-inflammatory molecules associated with MS. A cross-sectional study was carried out, including 95 patients with MS and 61 age- and gender-matched healthy controls (HCs). The enzyme-linked immunosorbent assay was used to detect anti-NR2 antibodies in serum samples of participants along with IgG antibodies against factor (F)VIIa, thrombin, prothrombin, FXa, and plasmin. According to our results, significantly elevated levels of anti-NR2 antibodies were detected in MS patients compared to HCs (p < 0.05), and this holds true when we compared the Relapsing-Remitting MS course with HCs (p < 0.05). A monotonically increasing correlation was found between NR2 seropositivity and advanced disability (r_s = 0.30; p < 0.01), anti-NR2 antibodies and disease worsening (r_s = 0.24; p < 0.05), as well as between antibody activity against NR2 and thrombin (r_s = 0.33; p < 0.01). The presence of anti-NR2 antibodies in MS patients was less associated with anti-plasmin IgG antibodies [OR:0.96 (95%CI: 0.92-0.99); p < 0.05]; however, such an association was not demonstrated when analyzing only RRMS patients. In view of our findings, NR2-reactive antibodies may play, paving the way for further research into their potential as biomarkers and therapeutic targets in MS.

Keywords: N-methyl-D-aspartate (NMDA) receptor; anti-coagulant serine proteases; antibodies; excitotoxicity; multiple sclerosis.

Figure 1

Distribution of antibodies against the NR2 subunit of the NMDAR between MS patients and healthy controls. The antibody activity was expressed as the ratio of a sample’s O.D. to the O.D. of the negative control. Mann-Whitney test applied for the analysis (* p < 0.05). AUC was measured for accuracy of the test MS: Multiple Sclerosis; NMDAR: N-Methyl-D-aspartate receptor; AUC: area under the curve.

Figure 2

Immunoglobulin activity against the NR2 subunit of the NMDAR between RRMS patients and healthy controls. The antibody activity was expressed as the ratio of a sample’s O.D. to the O.D. of the negative control. Mann-Whitney test applied for the analysis (* p < 0.05). AUC was measured for accuracy of the test. RRMS: Relapsing-Remitting Multiple Sclerosis; NMDAR: N-Methyl-D-aspartate receptor; AUC: area under the curve.

Figure 3

Comparative prevalence (%) of IgG detection in MS patients and HCs. Results are expressed as the percentage of participants tested positive (bars shown in black) or negative (bars shown in white) for the presence of anti-NR2 antibodies. Seropositivity was determined as follows: when the ratio was equal to or greater than 2.1, the sample was considered positive; and when the index was less than 2.1, the sample was considered negative.

Figure 4

Correlation between anti-NR2 antibodies and antibody activity against coagulant components. Positive correlation was observed between anti-NR2 and anti-FXa activity levels (A) and between anti-NR2 and anti-thrombin IgG (B). Negative correlation was demonstrated between anti-NR2 and anti-FVIIa IgG levels (C). The Spearman correlation coefficient was applied for the analysis. Values are expressed as the ratio of a sample’s O.D. to the O.D. of the negative control. Data with statistical significance are shown.

Figure 5

Binding activity against the NR2 subunit of the NMDAR based on the receiving medication for MS treatment. A significant difference was observed when assessing the ratio of NR2-reactive antibody values from patients receiving immunosuppressive medication with those obtained from HCs. Bars represent the mean ± SEM. The Kruskal-Wallis test was performed for the analysis and Dunn’s test for the multiple comparisons (* p < 0.05). MS: Multiple Sclerosis; IM: Immunomodulatory medication; IS: Immunosuppressive medication; HCs: healthy controls.