Investigating humoral autoimmunity against the NMDA-receptor NR1 subunit by super-resolution fluorescence microscopy: effects on synapse integrity and function
The project aims at unraveling structural and functional interrelations in the pathomechanism of antineuronal autoimmunity in the central nervous system (CNS) mediated by high-affinity autoantibodies (aAB). We focus on investigating the influence of purified pathogenic human IgG with specific aAB to the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor (anti-NR1 aAB) on the morphological integrity and function of the NMDA receptor ion channel, neuronal glutamatergic synapses, and intact neuronal networks. We apply single-molecule localization based super-resolution fluorescence imaging (direct stochastic reconstruction microscopy (dSTORM) and photoactivated localization microscopy (PALM)) combined with electrophysiological techniques, as well as 2-photon fluorescence imaging. We will use transfected HEK293 cells for outside-out patch recording with fast application of glutamate to evaluate anti-NR1 aAB-induced changes in biophysical properties of NMDA receptor ion channel function. For fluorescence application various labeling schemes for NMDA receptors and pathogenic aAB will be established including genetic marking and direct tagging with fluorophores. Super-resolution microscopy will be applied to quantify NMDA receptor subunits, subunit composition, and their interaction with anti-NR1 aAB.
In primary hippocampal neurons, we will combine single-bouton patch-clamp recordings with quantitative dSTORM microscopy to extract information about the molecular organization of NMDA receptors in postsynaptic terminals and extrasynaptic regions. In vital neurons, anti-NR1 aAB-induced changes in synaptic plasticity will be evaluated using live-cell dSTORM imaging of dendritic spines following long-term potentiation. Next, pathomechanisms of human anti-NR1 aAB will be investigated in the intact organism after intracerebral stereotactic application in mice. Here, we will evaluate the effects of anti-NR1 aAB on NMDA-receptor mediated glutamatergic transmission and short- and long-term synaptic plasticity. We will further establish an animal model of NMDA-receptor encephalitis by continuous delivery of pathogenic anti-NR1 aAB into the cerebrospinal fluid. Animals will be investigated for behavioral changes, as well as for disturbance of synaptic plasticity and hippocampal network activity by in vivo neurophysiology and two-photon imaging. Morphological changes will be investigated in brain slices by super-resolution fluorescence microscopy, thus improving current localization-based super-resolution microscopy methods for applications in tissue imaging. Together, in a proof-of-concept strategy we will elucidate the underlying pathomechanisms of anti-NMDA-receptor induced autoimmunity on the single molecule level with so far unmatched spatial and temporal resolution as a representative for a group of newly discovered CNS autoimmune disorders.
Sören Doose will coordinate and supervise the project together with other members of the team.
Christian Geis will coordinate and supervise the project together with other members of the team.
Dr. Werner, Christian
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