A05 - The Role of the RIM-binding protein AZ scaffold in regulating neurotransmitter release at diverse central synapses

Principal Investigators

Prof. Dr. Christian Rosenmund, Charité

Prof. Dr. Dietmar Schmitz, Charité

Presynaptic function is characterized by the tight temporal synchronization of an incoming action potential (AP) to the calcium-induced fusion of synaptic vesicles with the plasma membrane. A highly specialized protein scaffold at the active zone (AZ) is thought to enable the required close spatial proximity between voltage-gated calcium channels and the release machinery.

In project A05 we are investigating the role of RIM-binding proteins within this protein scaffold  (Figure 1) and their contribution to fast and synchronous vesicle fusion. Recent work on Drosophila RIM-binding protein (DRBP) has shown that DRBP is essential for the integrity of the AZ scaffold and required for efficient neurotransmitter release. The disruption of the single DRBP gene results in a defect in calcium channel clustering and calcium influx to the presynapse, which ultimately leads to a strong reduction of evoked release (Liu et al., 2011).

We are systematically investigating the role of RIM-BP1 and RIM‑BP2 in presynaptic function in murine central synapses using novel knockout mice for RIM-BP1 and RIM‑BP2. We are combining electrophysiological methods in neuronal culture and acute brain slices (Figure 2) with imaging techniques, such as high-pressure freezing electron microscopy (EM), Immuno-EM and super-resolution microscopy (STED) to dissect the function of RIM-BPs in molecular priming of synaptic vesicles and positional priming, i.e. the spatial coupling of calcium channels with release sites. Additionally, we will be doing detailed structure-function analysis to uncover the role of protein-protein interactions within the AZ scaffold.
 

Figure 1 Model of RIM-BP interactions Via their SH3 domains, RIM-BPs can simultaneously bind to RIMs, Calcium channels and the large scaffolding proteins Bassoon or Piccolo. RIMs interact also directly with presynaptic Calcium channels via their PDZ (postsynaptic density protein (PSD95), Drosophila Discs-large, epithelial tight junction protein ZO-1) domain and additionally bind to other active zone proteins, such as Munc13s and the SV-associated Rab3 family of small GTPbinding proteins.

 
 

Figure 2 Basic electrophysiological characterization of RIM-BP2 knockout neurons in autaptic hippocampal slices (A-C) and acute hippocampal slices (D-E). A Representative average traces of EPSCs evoked by brief 2 ms somatic depolarization to 0 mV (left) and summary graph of normalized EPSC amplitudes (right). Arrows indicate AP induction. (p = 0.0408, Mann-Whitney test). B Left: Representative traces (left) and summary graph (right) of synaptic responses to the application of hypertonic sucrose (500 mM) solution. C Summary graph of relative amplitudes in response to 5 APs triggered at 50 Hz. (n = 83 for WT and KO). D Representative traces (left) and summary graph (right) of field excitatory postsynaptic potentials (fEPSPs) in the stratum pyramidale in response to a paired pulse with indicated ISI.

References:

  • Arancillo, M., Min, S. W, Gerber, S., Munster-Wandowski, A., Wu, Y. J., Herman, M., Trimbuch, T., Rah, J. C., Ahnert-Hilger, G., Riedel, D., Sudhof, T. C., and Rosenmund, C.. "Titration of Syntaxin1 in mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability." J Neurosci 33(42): 16698-16714. (2013)

  • Beed, P., Gundlfinger, A., Schneiderbauer, S., Song, J., Bohm, C., Burgalossi, A., Brecht, M., Vida, I, and Schmitz, D.. "Inhibitory gradient along the dorsoventral axis in the medial entorhinal cortex." Neuron 79(6): 1197-1207 (2013).

  • Herman, M. A., Ackermann, F., Trimbuch, T., and Rosenmund, C. "Vesicular glutamate transporter expression level affects synaptic vesicle release probability at hippocampal synapses in culture." J Neurosci 34(35): 11781-11791 (2014).

  • Kintscher, M., Wozny, C., Johenning, F. W., Schmitz, D., and Breustedt, J.. "Role of RIM1alpha in short- and long-term synaptic plasticity at cerebellar parallel fibres." Nat Commun 4: 2392 (2013).

  • Kononenko, N. L., Diril, M. K., Puchkov, D., Kintscher, M., Koo, S. J., Pfuhl, G., Winter, Y., Wienisch, M., Klingauf, J., Breustedt, J, Schmitz, D., Maritzen, T., and Haucke, V. "Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2." Proc Natl Acad Sci U S A 110(6): E526-535 (2013).

  • Liu, K. S., Siebert, M., Mertel, S., Knoche, E., Wegener, S., Wichmann, C., Matkovic, T., Muhammad, K., Depner, H., Mettke, C., Buckers, J., Hell, S. W., Muller, M., Davis, G. W., Schmitz, D., and Sigrist, S. J. "RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release." Science 334(6062): 1565-1569 (2011).

  • Pangalos, M. , Donoso, J. R., Winterer, J., Zivkovic, A. R., Kempter, R., Maier, N., Schmitz D.. Recruitment of oriens-lacunosum-moleculare interneurons during hippocampal ripples. PNAS 110(11): 4398-403 (2013).

  • Trimbuch, T., Beed, P., Vogt, J., Schuchmann, S., Maier, N., Kintscher, M., Breustedt, J., Schuelke, M., Streu, N., Kieselmann, O., Brunk, I., Laube, G., Strauss, U., Battefeld, A., Wende, H., Birchmeier, C., Wiese, S., Sendtner, M., Kawabe, H., Kishimoto-Suga, M., Brose, N., Baumgart, J., Geist, B., Aoki, J., Savaskan, N. E., Brauer, A. U., Chun, J., Ninnemann, O., Schmitz, D., and Nitsch, R. "Synaptic PRG-1 modulates excitatory transmission via lipid phosphate-mediated signaling." Cell 138(6): 1222-1235 (2009).

  • Watanabe, S., Trimbuch, T., Camacho-Perez, M., Rost, B. R., Brokowski, B., Sohl-Kielczynski, B., Felies, A., Davis, M. W., Rosenmund, C., and Jorgensen, E. M. "Clathrin regenerates synaptic vesicles from endosomes." Nature 515(7526): 228-233 (2014).

  • Xue, M., Craig, T. K., Xu, J., Chao, H. T., Rizo, J., and Rosenmund, C. "Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity." Nat Struct Mol Biol 17(5): 568-575 (2010).