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. Volker Haucke
Institut für Chemie-Biochemie
Freie Universität Berlin
Takustr. 6
14195 Berlin
vhaucke@chemie.fu-berlin.de

Title

Activity-dependent regulation of pre- and postsynaptic membrane trafficking events

Question of the project

What is the role of synapsin in synaptic vesicle (re)clustering and how does this relate to the regulation of presynaptic plasticity? How do motor proteins contribute to the activity-dependent redistribution of endosomes near or into dendritic spines during long-term potentiation?

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). Over the past few years it has become clear that activity-dependent regulation of membrane trafficking pathways play a key role in these processes at both the cellular and molecular levels. Changes in the efficacy of excitation-secretion coupling or the synaptic vesicle cycle per se have been linked to presynaptic plasticity phenomena including PTP. Mice lacking the presynaptic vesicle-associated phosphoproteins synapsin I/II exhibit decreased PTP and severe synaptic depression upon repetitive stimulation (Rosahl et al., 1995) and this may relate to the activity-dependent phosphorylation of the synapsin A domain (Fiumara et al., 2007). The precise mechanistic basis for the role of synapsins in short-term plasticity is unclear but likely involves associations with the actin cytoskeleton and perhaps endocytic SH3 domain containing factors (see below). Unraveling the cellular and molecular mechanisms of synapsin-mediated PTP will thus be key to our understanding of short-presynaptic plasticity and the modulation of neurotransmitter release by activity.

A pivotal role of membrane traffic in synaptic plasticity has also been unravelled for postsynaptic LTP and LTD. Exocytic trafficking from recycling endosomes is required for increases in AMPA receptor cell surface number and for plasticity-induced growth of dendritic spines (Park et al., 2007). Conversely, the recognition of a basic endocytic sorting signals within the cytoplasmic domain of GluR1-3 by the mu subunit of the AP-2 adaptor complex underlies the internalization of cell surface AMPA receptors during LTD (Kastning et al., 2007). Whereas considerable progress has been made towards identifying the basic mechanisms of targeting postsynaptic receptors for activity-regulated exo-endocytosis, comparably little is known as to how the spatiotemporal dynamics of the corresponding endosomal organelles and their interactions with motor proteins and the cytoskeleton are regulated. These questions will thus be tackled in the forthcoming funding period.

Sheng M, Kim MJ (2002) Postsynaptic signaling and plasticity mechanisms. Science 298:776-780.
Fiumara F et al. (2007) Phosphorylation of synapsin domain A is required for post-tetanic potentiation. J Cell Sci 120:3228-3237.
Kastning K et al. (2007) Molecular determinants for the interaction between AMPA receptors and the clathrin adaptor complex AP-2. Proc Natl Acad Sci USA 104:2991-2996.
Park M et al. (2006) Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes. Neuron 52:817-830.
Rosahl TW et al. (1995) Essential functions of synapsins I and II in synaptic vesicle regulation. Nature 375:488-493.

Previous Work of the Group

Previous work in this laboratory has focussed on the identification of the mechanism by which pre- (Diril et al., 2006; Jung et al., 2007) and postsynaptic membrane proteins including neurotransmitter-gated ion channels are targeted for clathrin/AP-2-dependent endocytic internalization (Jung & Haucke, 2007) and recycling (Galli & Haucke, 2004). Moreover, we have begun to study the role of synapsin and its SH3 domain containing binding partners in synaptic vesicle clustering, a key event defective in synapsin knockout mice that display impaired PTP. During the past funding period we could show that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with mu2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin by AP-2mu. Our data therefore suggest that common mechanisms regulate AP-2-dependent internalization of pre- and postsynaptic membrane proteins. These findings likely are of key importance to understanding the basic mechanims of hippocampal LTD. Moreover, we have identified a positive feedback loop consisting of endocytic cargo proteins recognized by AP-2 and the synaptic isoform of phosphatidylinositol 4-phosphate 5-kinase (PIPK Igamma) that may provide a specific pool of phosphatidylinositol 4,5-bisphosphate dedicated to clathrin/AP-2 dependent internalization of pre- and postsynaptic membrane proteins (Krauss et al., 2006) as well as for exocytosis (Milosevic et al., 2005). Finally, we have been able to track down a novel role of endocytic SH3 domain containing endocytic proteins in synapsin-mediated synaptic vesicle clustering. Manipulation of complex formation between synapsin and endocytic SH3 domain proteins including the scaffolding protein intersectin by biochemical or genetic means leads to a near complete loss of the synaptic vesicle cluster and a corresponding decrease of synapsin association with recycling vesicle membranes (Pechstein et al., in preparation). We thus hypothesize that synapsin-SH3 domain protein complexes are essential components of the machinery for synaptic vesicle clustering, defects of which may impair short presynaptic plasticity phenomena including PTP.

Diril MK, Wienisch M, Jung N, Klingauf J, Haucke V (2006). Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling. Dev Cell 10:233-244
Galli T, Haucke V (2004). Cycling of synaptic vesicles: how far? How fast! Sci STKE 2004, re19
Jung N, Haucke V (2007) Clathrin-mediated endocytosis at synapses. Traffic 8:1129-1136
Jung N et al. (2007) Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2. J Cell Biol 179:1497-1510.
Kastning K et al. (2007) Molecular determinants for the interaction between AMPA receptors and the clathrin adaptor complex AP-2. Proc Natl Acad Sci USA 104:2991-2996.
Krauss M, Kukhtina V, Pechstein A, Haucke V (2006) Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes. Proc Natl Acad Sci USA 103:11934-11939.
Milosevic I et al. (2005) Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells. J Neurosci 25:2557-2565.

Objectives

The aim of the proposed project is to mechanistically dissect the role of synapsin and its SH3 domain containing binding partners in synaptic vesicle clustering and its relevance for short-term synaptic plasticity in vivo. Furthermore, we aim to track down motor proteins involved in the activity-dependent redistribution of endosomes near or into dendritic spines during long-term potentiation.

Methods

Molecular biology/ genetics (incl. transgenic mice; site-directed mutagenesis; RNA-interference); biochemical (incl. subcellular fractionation; affinity chromatography; organelle proteomics; GTPase & motor protein activity assays); c ell biological (transfection of primary neurons & neuronal cell lines; protein transduction; spinning disc confocal live cell microscopy; FM dyes & labelled ligands/ tracers for exo-endocytic recycling assays; pharmacological tools (i.e. chemical LTP induction)

Dissertation topics

1. Synapsin-SH3 domain interactions in short-term plasticity:

biochemical analysis of SH3 domain binding defective mutant synapsin I by co-immunoprecipitation, affinity chromatography, & direct binding assays;
generation of synapsin I/II knockout mice (available via collaboration with Drs. Paul Greengard, Rockefeller Univ & Fabio Benfenati, Italian Institute of Technology) transgenically expressing wild-type or SH3 domain binding defective mutant synapsin I (transgenic rescue);
analysis of presynaptic ultrastructure in hippocampal slices derived from transgenic mice (in collaboration with Dr. Gudrun Ahnert-Hilger);
analysis of synaptic vesicle recycling in hippocampal neurons expressing wild-type or mutant synapsin I (in a double KO background) using synaptophluorin as a tracer;
electrophysiological recordings to assess presynaptic short-term plasticity including PTP (in collaboration with other groups within the program).

2. Identification of motor proteins involved in synaptic plasticity:

affinity chromatography of proteins specifically associating with Rab11-GTP or Rab5-GTP following glycine stimulation
screen for candidate motor protein encoding genes upregulated by paradigms of synaptic plasticity, i.e. following high frequency stimulation or kainate application using Affymetrix gene chip technology (in collaboration with other members of the program);
analysis of the localization of candidate motor proteins tagged with eGFP in transfected primary neurons at rest or following electrical or chemical stimulation ("chemical LTP");
siRNA mediated knockdown of candidate motor proteins to assess effects on spine morphology, plasticity-induced spine growth & endosomal membrane dynamics (i.e. via Rab-eGFP);
localization and dynamics of AMPA and NMDA receptor containing organelles and their activity-dependent trafficking using live cell imaging approaches;
electrophysiological recordings from lentivirally transduced slice preparations (in collaboration with other groups within the program)

3. Functional proteomic characterization of AMPA receptor containing endosomal organelles

immunoisolation of AMPA receptor containing endosomal organelles from primary neurons labeled with ferromagnetic transferrin (two-step procedure);
MALDI MS/MS analysis of isolated organelles with emphasis on motor proteins & cytoskeleton;
biochemical and cell biological characterization of candidate proteins using conventional biochemical (binding assays, SPR) and imaging tools (confocal microscopy etc);
functional analysis (i.e. localization of AMPA receptor containing endosomal organelles) of candidate proteins using dominant negative & RNA interference approaches;
electrophysiological recordings (in collaboration with other groups within the program)

Cooperation with other Members

AG Ahnert-Hilger: Presynaptic plasticity mechanisms, electron microscopy analysis
AG Schmitz/Brecht: Plasticity phenomena in hippocampal slice preparations
AG Multhaup: D efective LTP in APP transgenic mice
AG Heinemann / Kempter: Electrophysiological recordings from slice preparations
AG Sigrist: STED microscopy analysis of synapsin mutant mice
AG Rosenmund: Analysis of presynaptic plasticity in autaptic cultures of hippocampal neurons

Scholarship Holder:

Arndt Pechstein (April 2005-March 2008)
Jelena Bacetic (since January 2008)

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