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. We previously demonstrated that a human GluN1 monoclonal antibody 5F5 (GluN1 mAb 5F5) rapidly localizes to and regulates synaptic NMDAR function at native synapses of primary hippocampal neurons. Here, we sought to explore signaling targets of GluN1 mAb 5F5 in primary cortical neurons of either sex using subcellular fractionation, Western blotting, and label-free quantitative phosphoproteomics by mass spectrometry. We find that human GluN1 mAb 5F5 does not change NMDAR abundance or surface levels of GluN1 on primary cortical neurons at 2 hr. Despite this, we observe that GluN1 mAb 5F5 alters the phosphoproteome in synaptoneurosomes and regulates numerous synapse-related biological processes and protein kinase activities. Bioinformatic analyses suggest that these phosphoproteomic 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. These studies may help to identify novel therapeutic strategies for anti-NMDAR encephalitis and other antibody-mediated encephalitides targeting cell surface antigens.
Joo, Y., Ciryam, P., Zhang, T., Huang, W., Dean, C. A., Lawrence, A. P., Vu, T. H., Gooya, N., Dessain, S. K., Kane, M. A., Benavides, D. R.
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