Abstract
N-Methyl-D-aspartate (NMDA) receptors (NMDARs) are among the most important excitatory neurotransmitter receptors in the human brain. Autoantibodies to the human NMDAR cause the most frequent form of autoimmune encephalitis involving autoantibody-mediated receptor cross-linking and subsequent internalization of the antibody-receptor complex. This has been deemed to represent the predominant antibody effector mechanism depleting the NMDAR from the synaptic and extra-synaptic neuronal cell membrane. To assess in detail the molecular mechanisms of autoantibody-induced NMDAR endocytosis, vesicular trafficking, and exocytosis we transiently co-expressed rat GluN1-1a-EGFP and GluN2B-ECFP alone or together with scaffolding postsynaptic density protein 95 (PSD-95), wild-type (WT), or dominant-negative (DN) mutant Ras-related in brain (RAB) proteins (RAB5WT, RAB5DN, RAB11WT, RAB11DN) in HEK 293T cells. The cells were incubated with a pH-rhodamine-labeled human recombinant monoclonal GluN1 IgG1 autoantibody (GluN1-aAbpH−rhod) genetically engineered from clonally expanded intrathecal plasma cells from a patient with anti-NMDAR encephalitis, and the pH-rhodamine fluorescence was tracked over time. We show that due to the acidic luminal pH, internalization of the NMDAR-autoantibody complex into endosomes and lysosomes increases the pH-rhodamine fluorescence. The increase in fluorescence allows for mechanistic assessment of endocytosis, vesicular trafficking in these vesicular compartments, and exocytosis of the NMDAR-autoantibody complex under steady state conditions. Using this method, we demonstrate a role for PSD-95 in stabilization of NMDARs in the cell membrane in the presence of GluN1-aAbpH−rhod, while RAB proteins did not exert a significant effect on vertical trafficking of the internalized NMDAR autoantibody complex in this heterologous expression system. This novel assay allows to unravel molecular mechanisms of autoantibody-induced receptor internalization and to study novel small-scale specific molecular-based therapies for autoimmune encephalitis syndromes.
Highlights
Most of the glutamatergic signaling mechanisms in the central nervous system (CNS) rely on the binding of this neurotransmitter (NT) to specific glutamate receptors (GluRs)
We surmised that the pH-rhodamine fluorescence is increased during the ensuing internalization of the N-Methyl-D-aspartate receptor (NMDAR)-autoantibody complex, due to the acidic luminal pH of endosomes and lysosomes
This might allow for mechanistic assessment of endocytosis, vesicular trafficking in both vesicular compartments and exocytosis of the NMDAR-autoantibody complex
Summary
Most of the glutamatergic signaling mechanisms in the central nervous system (CNS) rely on the binding of this neurotransmitter (NT) to specific glutamate receptors (GluRs). Ionotropic ligand-gated ion channels (iGluRs) and metabotropic G protein-coupled receptors (mGluRs) mediate fast and slow glutamatergic excitatory synaptic transmission at synapses between neuronal axons and dendrites [1]. Thereby, NMDARs serve a crucial function in synaptic plasticity (expressed as a change in receptor number and functional properties), learning, and memory. These processes start with the release of glutamate from presynaptic axon terminals and the subsequent binding together with the coagonist glycine mainly to postsynaptic NMDARs. Postsynaptic NMDARs, in turn, are associated with and regulated by several proteins that together constitute the postsynaptic density (PSD), an elaborate complex of interlinked proteins and elements of the cytoskeleton
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