Investigating humoral autoimmunity against the NMDA receptor GluN1 subunit by super-resolution fluorescence microscopy: effects on synapse integrity and function

NMDAR(NMDAR)的GluN1亚基是引起NMDAR脑炎的致病自身抗体的靶位。在之前的工作中，我们已经建立了超分辨率成像技术和图像处理工具，用于初级神经元和脑组织中受体的可视化。通过使用这些先进的成像工具和电生理学，我们发现患者来源的GluN1抗体减少了突触后受体区域中NMDAR的表达，并减少了单个突触中NMDAR介导的电流。这些变化是针对GluN1抗体的，因为我们使用了患者来源的纯化的GluN1抗体和人类单抗GluN1。在最初的初步研究中，我们通过插入非天然氨基酸和点击化学方法成功地将GluN1亚基的生物正交标记应用于HEK293细胞中表达的功能性NMDAR。此外，我们应用了一种新的NMDAR脑炎小鼠模型，持续脑室内被动转移致病抗体，发现类似的模式，膜结合的NMDAR下调，海马区突触可塑性缺陷，并严重降低记忆能力。我们现在将(I)解决新的方法工具来可视化与疾病相关的抗体-受体的直接相互作用，以及(Ii)确定抗NMDAR抗体介导的突触可塑性障碍的关键事件，以确定特定治疗方法的可能性。NMDAR在神经元和重组GluN1抗体中的点击化学使固定神经元的超分辨率成像以及活体成像和单分子跟踪实验能够进行特定、高灵敏度和定量的标记。结合细胞外，活细胞，点击化学标记和3D晶格光片显微镜，将被用来确定抗体结合的NMDAR的细胞内转运途径。为了研究NMDAR介导的可塑性及其对人类致病抗体的干扰，我们将在活体神经元中进行超分辨时间推移成像，并结合膜片钳电生理学。我们将根据突触电流和突触长时程增强的功能分析来确定突触后脊椎形态和受体定位的变化。此外，我们将探讨NMDAR自身免疫如何影响钙/钙调蛋白依赖的蛋白激酶II(CaMKII)的作用，CaMKII是NMDAR的直接相互作用伙伴和突触可塑性的关键分子。最后，在概念验证的方法中，我们将确定在NMDAR脑炎小鼠模型中，是否可以通过抑制突触CaMKII的对应物DAPK1在体外和体内挽救突触的可塑性。这些实验将使我们能够以迄今为止无与伦比的灵敏度和准确性剖析NMDAR信号和病理，这可能导致识别NMDAR脑炎的创新干预策略。

The GluN1 subunit of the NMDA receptor (NMDAR) is the target epitope for pathogenic autoantibodies causing NMDAR encephalitis. In previous work, we have established super-resolution imaging techniques and image-processing tools for receptor visualization in primary neurons and in brain tissue. By using these advanced imaging tools together with electrophysiology we showed that patient-derived GluN1 antibodies reduce NMDAR expression in postsynaptic receptor fields along with decrease of NMDAR mediated current in individual synapses. These changes were specific for antibodies to GluN1 as we used patients´ derived purified GluN1 IgG and human monoclonal GluN1 antibodies. In first pilot studies we successfully applied bioorthogonal labeling of the GluN1 subunit by insertion of unnatural amino acids and click chemistry in functional NMDAR expressed in HEK293 cells. Furthermore, we applied a novel mouse model of NMDAR encephalitis with continuous intraventricular passive-transfer of pathogenic antibodies and found a similar pattern of downregulation of membrane-bound NMDAR, defective synaptic plasticity in the hippocampus, and severely reduced memory performance. We will now (i) address novel methodical tools to visualize direct disease-relevant antibody-receptor interactions and (ii) identify key events of anti-NMDAR antibody-mediated dysfunction of synaptic plasticity to identify possibilities for specific therapeutic approaches. Click chemistry of NMDAR in neurons and in recombinant GluN1 antibodies enables a specific, highly sensitive and quantitative labeling for super-resolution imaging of fixed neurons as well as for intravital imaging and single-molecule tracking experiments. Combination of extracellular, live-cell labeling by click chemistry and 3D lattice-light sheet microscopy will be used to determine the intracellular trafficking pathways of antibody-bound NMDARs. To investigate NMDAR mediated plasticity and their interference by human pathogenic antibodies we will perform super-resolved time-lapse imaging in vital neurons together with patch-clamp electrophysiology. We will determine changes in postsynaptic spine morphology and receptor localization with respect to functional analyses of synaptic currents and synaptic long-term potentiation. Furthermore, we will explore how NMDAR autoimmunity affects the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a direct NMDAR interaction partner and key molecule of synaptic plasticity. Finally, in a proof-of-concept approach, we will determine if synaptic plasticity can be rescued by inhibition of the synaptic CaMKII counterpart DAPK1 in-vitro and in-vivo in our mouse model of NMDAR encephalitis. These experiments will allow us to dissect NMDAR signaling and pathology with so far unmatched sensitivity and accuracy which possibly leads to identification of innovative intervention strategies in NMDAR encephalitis.