An Assay to Determine Mechanisms of Rapid Autoantibody-Induced Neurotransmitter Receptor Endocytosis and Vesicular Trafficking in Autoimmune Encephalitis

Frontiers in Neurology

Christoph Brenker¹  Timo Strünker¹  Elsie Amedonu²  Sven G. Meuth²  Heinz Wiendl²  Andre Dik²  Christine Strippel²  Nico Melzer²  Hans-Peter Hartung³  Norbert Goebels³  Sumanta Barman³  Bernhard Wünsch⁴  Thomas Budde⁵  Nathalie Strutz-Seebohm⁶  Stefan Peischard⁶  Guiscard Seebohm⁶  Julian A. Schreiber⁶  Sebastian Becker⁶ 

[1] Centre of Reproductive Medicine and Andrology, University of Muenster, Muenster, Germany;Department of Neurology, University of Muenster, Muenster, Germany;Department of Neurology, Universitätsklinikum and Center for Neurology and Neuropsychiatry LVR Klinikum, Heinrich Heine University Duesseldorf, Duesseldorf, Germany;Institute for Pharmaceutical and Medical Chemistry, University of Muenster, Muenster, Germany;Institute for Physiology I, University of Muenster, Muenster, Germany;Myocellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases, University of Muenster, Muenster, Germany;

关键词: autoimmune encephalitis;    N-Methyl-D-aspartate receptors;    cross-linking;    endocytosis;    vesicular trafficking;    exocytosis;   

DOI  :  10.3389/fneur.2019.00178

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown