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Chemical synapses are major sites of communication between neurons. Synapses are often formed by an axon terminal of the presynaptic neuron and a dendritic spine of the postsynaptic cell (Fig. 1). The postsynaptic density (PSD) is a cytoskeletal specialization beneath the postsynaptic membrane. This protein network assembles neurotransmitter receptors, associated signalling proteins, and scaffolding proteins to organize the signal transduction pathways near the postsynaptic membrane. The protein composition of PSDs is dynamically regulated by neuronal activity. This is thought to be the major molecular basis for synaptic plasticity.
Using a proteomics approach, the project aims at the identification of components of biochemical fractions enriched in synaptic (notably PSD) proteins and at the functional characterization of such proteins (with particular emphasis on Neuroplastins) in different models of synaptic plasticity.
(1) Protein composition of synaptic structures
K.-H. Smalla, K. Pohlmann, W. Tischmeyer
Identification of synaptic proteins
In collaboration with K.W. Li and A.B. Smit (Amsterdam), and E.D. Gundelfinger (LIN) the proteome of a PSD-enriched fraction from rat brain has been investigated using two-dimensional gel electrophoretic separation (Fig. 2) in conjunction with mass spectrometric identification of proteins (Li et al 2004). An isotope-coded affinity tag (ICAT) based correlation profiling approach was used to confirm the enrichment of well-established core PSD proteins in the PSD fraction as compared to proteins of a synaptic membrane fraction (Li et al 2005). Other functional groups of proteins – such as cytoskeleton-associated proteins, protein kinases and phosphatases, components and regulators of signaling pathways, and proteins involved in energy production – associate with multiple organelles and, consequently, were enriched in the PSD fraction to a lesser extent.
To support research focussing on synaptic proteins and their interactions, the database SynProt (Pielot et al 2012) was compiled in collaboration with E.D. Gundelfinger (LIN) and D.C. Dieterich (OvGU). This database compiles the published data from proteomic studies on the protein composition of PSDs.
Dynamics of synaptic proteins
In an animal model for temporal lobe epilepsy, changes in the protein composition of a PSD-enriched fraction were studied in collaboration with U. Wyneken (Santiago de Chile). After extensive synaptic activity associated with seizures, we monitored significant changes in the abundance of different receptors, signaling enzymes and cytoskeletal proteins. Electrophysiological recordings from PSDs re-incorporated into giant liposomes suggest that such synaptic rearrangements have profound consequences on the function of the NMDA-type glutamate receptor (Wyneken et al 2001).
In a corporate approach within the SFB 779 project, changes in synaptic protein composition after auditory discrimination learning were investigated in different brain regions of mice (Kähne et al 2012). Most notably, the majority of variations in protein composition are for cytoskeletal and scaffold proteins. This suggests that learning induces plastic reorganizations of synaptic cytoskeletal matrices.
Our recent work is focused on the analysis of synaptic fractions from transgenic mice to identify gene-related proteome changes in various paradigms of synaptic plasticity. Affinity-purified protein complexes of synaptic fractions are used to characterize in particular specific protein-protein interactions. These investigations are based on subcellular fractionation techniques, affinity chromatography, 2D gel electrophoresis, high-resolution chromatography (HPLC, ÄKTA, FPLC) and identification of proteins by immunoaffinity and mass spectrometry techniques.
Kähne T, Kolodziej A, Smalla KH, Eisenschmidt E, Haus UU, Weismantel R, Kropf S, Wetzel W, Ohl F, Tischmeyer W, Naumann M, Gundelfinger ED (2012). Synaptic proteome changes in mouse brain regions upon auditory discrimination learning. Proteomics 12(15-16):2433-44. Epub 2012 Jun 14
Li KW, Hornshaw MP, Smalla KH, Gundelfinger ED, Smit AB (2005). Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins. J Proteom Res 4:725-33.
Pielot R, Smalla KH, Müller A, Landgraf P, Lehmann AC, Eisenschmidt E, Haus UU, Weismantel R, Gundelfinger ED, Dieterich DC (2012) SynProt: A Database for Proteins of Detergent-resistant Synaptic Protein Preparations. Frontiers in Synaptic Neuroscience 2012, published: 25 June 2012, doi: 10.3389/fnsyn.2012.00001
Wyneken, U., Smalla, K.H., Marengo, J.J., Soto, D., de la Cerda, A, Tischmeyer, W., Grimm, R., Boeckers, T.M., Wolf, G., Orrego, F. and Gundelfinger, E.D. (2001). Kainate-induced seizures alter protein composition and N-methyl-D-aspartate receptor function of rat forebrain postsynaptic densities. Neuroscience 102:65-74.
(2) Investigation of Neuroplastin Interactomes
R. Herrera-Molina, K. Pohlmann, K.H. Smalla
Neuroplastin (NP) proteins are glycoproteins of the immunoglobulin (Ig) superfamily. Whereas the two-Ig domain isoform NP-55 is expressed in many tissues, the three-Ig domain isoform NP-65 is brain-specific, enriched in PSD protein preparations, and exhibits trans homophilic binding (Smalla et al 2000). Enhanced association of NP-65 to PSDs after strong synaptic activation (Smalla et al 2000) suggests activity-dependent plastic rearrangements in NP-65 containing protein complexes. Therefore, in order to gain more insight into the functions of NP proteins in processes of neuroplasticity, current work aims to characterise NP protein interaction networks. In a first study, we identified complexes containing NP and alpha and beta subunits of GABAA receptors (Sarto-Jackson et al 2012). Partial co-localisation of NP-65 with ß2/3-subunits of GABAA receptors in primary neuronal cultures is shown in Fig. 3.
Smalla, K.H. , Matthies, H., Langnaese, K., Boeckers, T.M. Wyneken, U., Staak, S., Krug, M., Beesley, P. W. and Gundelfinger, E.D. (2000) The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses. Proc. Natl. Acad. Sci. USA 97 , 4327-4332
Marzban H, Khandzada U, Shabir S, Hawkes R, Langnaese K, Smalla KH, Bockers TM, Gundelfinger ED, Gordon-Weeks PR and Beesley PW (2003) Expression of the Immunoglobulin Superfamily Neuroplastin Adhesion Molecules in Adult and Developing Mouse Cerebellum and Their Localisation to Parasagittal Stripes. J Comp Neurol 462: 286–301
Bernstein HG, Smalla KH, Bogerts B, Beesley PW, Gundelfinger ED, Kreutz, MR (2007) The immunolocalization of the synaptic glycoprotein neuroplastin differs substantially between the human and the rodent brain. Brain Res 1134(1):107-12. Epub 2006 Dec 27.
Sarto-Jackson I, Milenkovic I, Smalla KH, Gundelfinger ED, Kaehne T, Herrera-Molina R, Kiebler MA, Sieghart W (2012) The cell adhesion molecule neuroplastin-65 is a novel interaction partner of GABA-A receptors. J Biol Chem 287(17):14201-14. Epub 2012 Mar 2.
Mlinac K, MiloševiÄ‡ NJ, Heffer M, Smalla KH, Schnaar RL, Kalanj Bognar S (2012) Neuroplastin expression in the hippocampus of mice lacking complex gangliosides. J Mol Neurosci 48(1):161-6. Epub 2012 May 26