Department of Molecular Neuroscience, Graduate School of Innovative Life Science, University of Toyama, Toyama 930-0194, Japan.
Neurosci Res. 2011 Nov;71(3):294-302. doi: 10.1016/j.neures.2011.07.1834. Epub 2011 Aug 5.
The concept of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, a severe, potentially lethal, treatment-responsive disorder, mediated by autoantibodies against NMDAR was proposed. Because paraneoplastic anti-NMDAR encephalitis has a better prognosis after tumor resection and immunotherapy, rapid quantitative systems for detecting functional autoantibodies against extracellular epitopes of NMDAR are necessary. To detect autoantibodies recognizing extracellular epitopes of NMDAR, we stably expressed mutant NMDAR that decreases Ca(2+) permeability on a heterologous cell surface without any antagonist. Serum and CSF samples from patients were analysed using the cells expressing mutant NMDAR subunits by immunocytochemistry and on-cell Western analysis using live cells stably expressing mutant NMDAR. Furthermore, we were able to express mutant GluRζ1(NR1, GluN1) subunit of NMDAR alone on the cell surface and obtained direct evidence of the presence of autoantibodies recognizing extracellular epitopes of GluRζ1 and the induction of internalization by autoantibodies in serum and CSF from patients. The specificity of on-cell Western analysis was improved at 37°C. The combination of this rapid quantitative assay using our on-cell western analysis, detailed analysis of extracellular epitopes of NMDAR, and internalization assay of NMDAR will be valuable for the diagnosis, evaluation of clinical treatments, and follow-up of anti-NMDAR encephalitis.
抗 N-甲基-D-天冬氨酸受体（NMDAR）脑炎是一种严重的、潜在致命的、可治疗的疾病，其发病机制是针对 NMDAR 的自身抗体。由于肿瘤切除和免疫治疗后副肿瘤性抗 NMDAR 脑炎的预后较好，因此需要快速定量检测针对 NMDAR 细胞外表位的功能性自身抗体。为了检测识别 NMDAR 细胞外表位的自身抗体，我们在异源细胞表面稳定表达了突变型 NMDAR，该突变型 NMDAR 降低了 Ca(2+)通透性，且没有任何拮抗剂。通过免疫细胞化学和活细胞表面表达突变型 NMDAR 的 on-cell Western 分析，对来自患者的血清和 CSF 样本进行了分析。此外，我们能够在细胞表面单独表达突变型 GluRζ1（NR1，GluN1）亚基的 NMDAR，并获得了来自患者血清和 CSF 中识别 GluRζ1 细胞外表位的自身抗体和诱导内化的直接证据。在 37°C 时，on-cell Western 分析的特异性得到了提高。该快速定量测定法与我们的 on-cell Western 分析相结合，对 NMDAR 细胞外表位的详细分析以及 NMDAR 的内化测定法将有助于抗 NMDAR 脑炎的诊断、临床治疗评估和随访。
