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

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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.