Objective:

To explore signaling targets of a human GluN1 monoclonal antibody 5F5 (GluN1 mAb 5F5) in rat primary cortical neurons of either sex using biochemistry, subcellular fractionation, and label-free quantitative phosphoproteomics.

Background:

The most common form of autoimmune encephalitis is associated with antibodies that target N-methyl-D-aspartic acid receptors (NMDARs). NMDARs play a pivotal role in neurotransmission and synaptic plasticity. Mounting evidence has shown that antibody targeting of the NMDAR GluN1 subunit, as in anti-NMDAR encephalitis, leads to NMDAR cross-linking and receptor internalization. However, the underlying signaling pathways affected by antibodies targeting NMDARs remain to be explored. 

Design/Methods:

We employed subcellular fractionation of primary cortical neurons to generate synaptoneurosomes (SNs) and postsynaptic density (PSD) fractions. We isolated SNs or PSD fractions after GluN1 mAb or Control mAb exposure to explore changes in synaptic signaling pathways. This approach enabled us to focus on GluN1 antibody-mediated synaptic pathology. A quantitative phosphoproteomic analysis using mass spectrometry identified kinase signaling cascades regulated by GluN1 mAbs compared to Control mAb in SNs.

Results:

Phosphoproteomic analyses by mass spectrometry demonstrate that GluN1 mAb 5F5 alters protein phosphorylation in SN fractions and regulates numerous synapse-related biological processes and protein kinase activities. Bioinformatic analyses suggest that these phosphoproteomic proteomic changes are positively correlated with NMDAR activation and negatively correlated with NMDAR inhibition. Together, these data suggest that GluN1 mAb 5F5 alters intracellular kinase signaling pathways in primary cortical neurons, likely by activating the NMDAR. 

Conclusions:

The pathophysiology of anti-NMDAR encephalitis remains poorly defined but is currently thought to involve pathogenic antibodies that crosslink and internalize NMDARs. We identify intracellular signaling pathways targeted by human NMDAR antibodies in primary cortical neurons. Our key findings are obtained using specific, patient-derived human monoclonal antibodies with high affinity to the GluN1 subunit of NMDARs. These studies expand the underlying pathophysiological molecular mechanisms involved in anti-NMDAR encephalitis.