Investigating humoral autoimmunity against the NMDA-receptor NR1 subunit by super-resolution fluorescence microscopy: effects on synapse integrity and function

Project Description

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.

Title Year Authors Journal Links
Autoimmune seizures and epilepsy 2019 Geis, C., Planagumà, J., Carreño, M., Graus, F., and Dalmau, J.. J Clin Invest More
Human autoantibodies against the AMPA receptor subunit GluA2 induce receptor reorganization and memory dysfunction 2018 Haselmann, H., Mannara, F., Werner, C., Planagumà, J., Miguez-Cabello, F., Schmidl, L., Grünewald, B., Petit-Pedrol, M., Kirmse, K., Classen, J., Demir, F., Klöcker, N., Soto, D., Doose, S., Dalmau, J., Hallermann, S., and Geis, C. Neuron More
LGI1 antibodies alter Kv1.1 and AMPA receptors changing synaptic excitability, plasticity and memory 2018 Petit-Pedrol, M., Sell, J., Planaguma, J., Mannara, F., Radosevic, M., Haselmann, H., Ceanga, M., Sabater, M., Spatola, M., Soto, D., Gasull, X., Dalmau, J., and Geis, C. Brain More
Bioorthogonal click chemistry enables site-specific fluorescence labeling of functional NMDA receptors for super-resolution imaging 2018 Neubert, F., Beliu, G., Terpitz, U., Werner C., Geis, C., Sauer, M., and Doose, S. Angew Chem Int Ed More
Autoantibodies to synaptic receptors and neuronal cell surface proteins in autoimmune diseases of the central nervous system 2017 Dalmau, J., Geis, C., and Graus, F. Physiol Rev More
Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis 2017 Grünewald, B., Lange, M.D., Werner, C., O'Leary, A., Weishaupt, A., Popp, S., Pearce, D.A., Wiendl, H., Reif, A., Pape, H.C., Toyka, K.V., Sommer, C., and Geis, C. eLife More
Gephyrin-binding peptidesvisualize postsynaptic sites and modulate neurotranmission 2016 Maric, H. M., Hausrat, T. J., Neubert, F., Dalby, N. O., Doose, S., Sauer, M., Kneussel, M., and Stromgaard, K. Nat Chem Biol More
Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity 2016 Planagumà, J., Haselmann, H., Mannara, F., Petit-Pedrol, M., Grünewald, B., Aguilar, E., Röpke, L., Martín-García, E., Titulaer, M. J., Jercog, P., Graus, F., Maldonado, R., Geis, C., and Dalmau, J. Ann Neurol More
Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition 2016 Werner, C., Pauli, M., Doose, S., Weishaupt, A., Haselmann, H., Grunewald, B., Sauer, M., Heckmann, M., Toyka, K. V., Asan, E., Sommer, C., and Geis, C. Brain More
Stiff personsyndrome IgG affects presynaptic GABAergic release mechanisms 2014 Werner, C., Haselmann, H., Weishaupt, A., Toyka, K. V., Sommer, C., and Geis, C. J Neural Transm More
Super-resolution imaging of plasma membrane glycans 2014 Letschert, S., Göhler, A., Franke, C., Bertleff-Zieschang, N., Memmel, E., Doose, S., Seibel, J., and Sauer, M. Angew Chem More
Methylene Blue- and Thiol-Based Oxygen Depletion for Super-Resolution Imaging 2013 Schäfer, P., van de Linde, S., Lehmann, J., Sauer, M., and Doose, S. Anal Chem More
Human Stiff person syndrome IgG-containing high-titer anti-GAD65 autoantibodies induce motor dysfunction in rats 2013 Hansen, N., Grunewald, B., Weishaupt, A., Colaco, M. N., Toyka, K. V., Sommer, C., and Geis, C. Exp Neurol More
Human IgG directed against amphiphysin induces anxiety behavior in a rat model after intrathecal passive transfer 2012 Geis, C., Grunewald, B., Weishaupt, A., Wultsch, T., Toyka, K. V., Reif, A., and Sommer, C. J Neural Transm More
Human stiff-person syndrome IgG induces anxious behavior in rats 2011 Geis, C., Weishaupt, A., Grunewald, B., Wultsch, T., Reif, A., Gerlach, M., Dirkx, R., Solimena, M., Perani, D., Heckmann, M., Toyka, K. V., Folli, F., and Sommer, C. PLoS ONE More
Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition 2010 Geis, C., Weishaupt, A., Hallermann, S., Grunewald, B., Wessig, C., Wultsch, T., Reif, A., Byts, N., Beck, M., Jablonka, S., Boettger, M. K., Uceyler, N., Fouquet, W., Gerlach, M., Meinck, H. M., Siren, A. L., Sigrist, S. J., Toyka, K. V., Heckmann, M., and Sommer, C. Brain More
Stiff person syndrome associated anti-amphiphysin antibodies reduce GABA associated [Ca(2+)]i rise in embryonic motoneurons 2009 Geis, C., Beck, M., Jablonka, S., Weishaupt, A., Toyka, K. V., Sendtner, M., and Sommer, C. Neurobiol Dis More