Abstract Details

Title Protein Kinase Signaling as a Downstream Target of Human Anti-NMDA Receptor Antibodies

Topic Autoimmune Neurology

Presentation(s) C14 - Autoimmune Encephalitis: Pathophysiology to Treatment (11:01 AM-11:11 AM)
P3 - Poster Session 3 (11:30 AM-12:30 PM)

Poster/Presentation
Number 1-001

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.

Authors/Disclosures
David R. Benavides, MD, PhD, FAAN (University of Maryland School of Medicine) PRESENTER	The institution of Dr. Benavides has received research support from F. Hoffman La Roche Ltd.
Yuyoung Joo, PhD	Dr. Joo has nothing to disclose.
Prajwal Ciryam, MD, PhD (University of Maryland School of Medicine)	The institution of Dr. Ciryam has received research support from Henry M. Jackson Foundation. The institution of Dr. Ciryam has received research support from Passano Foundation. The institution of Dr. Ciryam has received research support from GEn1E Lifesciences. The institution of Dr. Ciryam has received research support from Brain Aneurysm Foundation. The institution of Dr. Ciryam has received research support from Neurocritical Care Foundation. The institution of Dr. Ciryam has received research support from Center for Shock, Trauma, and Anesthesiology Research, University of Maryland.
Timothy Zhang	Timothy Zhang has nothing to disclose.
Weiliang Huang	Weiliang Huang has nothing to disclose.
Charles Dean	Mr. Dean has nothing to disclose.
Audrey Lawrence	Ms. Lawrence has nothing to disclose.
Thanh Hien T. Vu	Ms. Vu has nothing to disclose.
Niki Gooya, PhD	Dr. Gooya has nothing to disclose.
Scott K. Dessain, MD, PhD	Dr. Dessain has nothing to disclose.
Maureen A. Kane, PhD	Prof. Kane has nothing to disclose.

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