GRK 1123 - Learning and Memory

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GRK 1123:

Cellular Mechanisms of Learning and Memory Consolidation
in the Hippocampal Formation

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This Research Training Group is funded by the German Research Council DFG

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AG Ahnert-Hilger	AG Behr	AG Geiger	AG Haucke	AG Heinemann/ Kempter
AG Multhaup	AG Wulczyn	AG Rosenmund	AG Schmitz/ Brecht	AG Sigrist

Prof. Dr. Christian Rosenmund
Neurowissenschaftliches Forschungszentrum
Charité Universitätsmedizin Berlin
Charitéplatz 1
10117 Berlin
christian.rosenmund@charite.de

Topic

Regulation of transmitter release

Title

Role of presynaptic proteins in regulating synaptic function and plasticity

Issues of the project

When trains of action potentials reach a nerve terminal, the synapse transduces the electrical signals into chemical signals via release of neurotransmitters, in turn activating postsynaptic receptors and altering the postsynaptic membrane potential. How this presynaptic transduction process works and how it shapes, and is shaped by the information it encodes, is not well understood. Critical parameters in synaptic transmission, such as speed of vesicular release, reliability and synaptic strength during ongoing activity, have different weights depending on whether a synapse transduces information about sound localization or is involved in memory formation. These underlying transduction processes are not hard wired, as synapses may change functional parameters as the brain develops, with activity, experience and during disease. The involved proteins are studied by reverse engineering approaches.

Current State of Research

Chemical neurotransmission in the central nervous system is under activity- and experience-dependent control that may involve both pre- and postsynaptic mechanisms. These include short-term presynaptic enhancement of neurotransmitter release as seen in posttetanic potentiation (PTP) as well as long-term changes in synaptic responses due to the modulation of the number and composition of postsynaptic ion channels during long-term potentiation (LTP) or depression (LTD) (Sheng & Kim, 2002). Apart from snare proteins a number of other snare associated proteins are involved in mediating synaptic transmitter relase. These include vesicular transporters for GABA and glutamate but also the Munc 13 family involved in priming of vesicles. Surprisingly, these molecules play also a key role in modulating presynaptic plasticity, as different isoforms mediate drastically different short-term plasticity behavior. Furthermore, two proteins are particularly been implicated in regulating Ca2+ triggered release: the candidate Ca2+-sensor of release, synaptotagmin I and the SNARE complex associated Complexins. Neurons lacking either of these proteins show a dramatic decrease in neurotransmitter release efficacy and a poorly timed release time course.

Sheng M, Kim MJ (2002) Postsynaptic signaling and plasticity mechanisms. Science 298:776-780.

Previous work of the group

The overall research questions in the laboratory are generally focussed on how synaptic vesicles get ready to become fusion competent, and how Ca2+ triggers the fusion event. We utilize known molecular components of synaptic function to study structure/function relationships and how the nervous system uses variations of protein composition. We are also identifying novels proteins and their functions in mammalian system to study presynaptic function during 1) the development of the nervous system, 2) short- and long term plasticity, and 3) in disease.

Essential to our current functional analysis of the presynapse is the single cell autaptic culture. This preparation allows us to easily record synaptic activity using standard electrophysiology while maintaining optimal control of the neuronal environment by rapid solution exchange. It also offers excellent optical access for performing quantitative imaging of axonal and synaptic structures and determining synaptic vesicle turnover activity using styryl dye uptake and other fluorescent-based imaging methods. To characterize the function of essential proteins in synaptic transmission, we use a combination of loss-of-function and gain-of-function-rescue approaches. We first use knockout mice to define the general function of a protein by associating the phenotype to a specific step in the release process. We then use lentiviral overexpression of wildtype and mutant versions in neurons from knockout mice. This allows an in-depth analysis of protein function to reveal the role of structurally or biochemically defined protein domains and protein-protein interactions. We are planning to use current mouse models and novel ones created in our laboratory to probe the impact of specific manipulations of synaptic function on neuronal networks and ultimately behavior. We have been particularly involved in characterizing the function of Munc 13 isoforms, of the versicular transporters, the synaptotagmin Ca sensor and the complexins.

At the postsynapse, we have worked over the years on several structure-function aspects of AMPA- and Kainate receptor, on the mechanism of AMPA receptor assembly, and the interaction of receptor subunits in gating. We are currently studying the molecular basis of heteromeric AMPA receptor assembly and gating. Furthermore, we use engineered AMPA receptors at the synapse to quantify the neurotransmitter concentration profile at the synapse, and how receptor desensitization affects the reliability of synapses.

Rosenmund, C., Sigler, A., Augustin, I., Reim, K., Brose, B. and Rhee, J.S. (2002). Differential Control of Vesicle Priming and Short-Term Plasticity by Munc13 Isoforms. Neuron 33, 411-425.
Wojcik, S.M., Rhee, J.S., Herzog, E., Sigler, A., Jahn, R., Takamori, S., Brose, N.* and Rosenmund, C. (2004). An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size. Proc. Nat. Acad. Sci. USA 101:7158-63.
Rosenmund, C., Rettig, J. and Brose, N. (2004) Molecular mechanisms of active zone function. Curr. Opin. Neurobiol. 13(5) 509-19.
Weston, M, Gertler, C., Mayer, M.L. and Rosenmund, C. (2006) Interdomain interactions in AMPA and kainate receptors regulate affinity for glutamate. J. Neurosci. 26(29):7650-8.
Basu, J., Betz, A., Brose, N., and Rosenmund, C. (2007) Munc13-1 C1 domain activation lowers the energy barrier for synaptic vesicle fusion. J. Neurosci. 27, 1200-1210.
Rizo, J. and Rosenmund, C. (2008) Synaptic Vesicle Fusion. Review. Nat. Struct. Mol. Biol. Jul;15(7):665-74.
Xue., M., Reim, K., Chen, X., Chao, H.T., Deng, H., Rizo, J., Brose, N. and Rosenmund, C. (2007) Distinct domains of complexin I differentially regulate neurotransmitter release. Nat. Struct. Mol. Biol. Oct 14(10):949-58.

Objectives

The aim of the proposed project is to mechanistically dissect the role of SNARE complex associated proteins in regulating transmitter release. By use of knock out animals and transfection with normal and mutant proteins we will study the function and interaction with other proteins and the role which different protein interaction sites play in regulating transmitter release.

Methods

Molecular biology/ genetics: transgenic mice; site-directed mutagenesis; RNA-interference, transfection techniques, mutation analysis
Electrophysiology: patch clamp recordings from cultured neurons, neurons from acute brain slices and from transfected cell lines.
Imaging: In vivo imaging from nerve terminals to study Ca dynamics ans synaptic vesicle turnover.
Morphology: Quantitative analysis of protein expression in native tissue, quantification of dendritic, axonal and synaptic structures in vitro and in vivo. Ultrastructural analysis of synaptic terminals using standard and cryo electron microscopy.

Topics for Thesis Projects

Complexin synaptotagmin interactions in regulating Ca triggered neurotransmitter release
Analysis of Munc 13 isoforms in reguating vesicle priming and synaptic plasticity
Analysis of vesicular transporter function and its impact on synaptic parameters.

Cooperation with other Members of the Graduate School

AG Ahnert-Hilger: Vesicular neurotransmitter transporter, presynaptic plasticity mechanisms, electron microscopy analysis
AG Schmitz/Brecht: Plasticity phenomena in hippocampal slice preparations
AG Heinemann/Kempter: Electrophysiological recordings from slice preparations
AG Haucke: Regulation of vesicle trafficking
AG Sigrist: Organization and function of the presynaptic active zone

Scholarship Holder:

Zimmermann, Johannes (since February 2010)

AG Ahnert-Hilger	AG Behr	AG Geiger	AG Haucke	AG Heinemann/ Kempter
AG Multhaup	AG Nitsch/ Wulczyn	AG Rosenmund	AG Schmitz/ Brecht	AG Sigrist