Nerve Cells Masters of Communication

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1 Nerve Cells Masters of Communication The human nerve system consists of approximately one hundred billion nerve cells give or take a few billions. And each one of these cells has the ability to make contact with thouands of neighboring cells. Tiny membrane bubbles, the so-called vesicles, set free special messengers that affect the behavior of adjacent cells. To study what happens during these processes in detail on the molecular level is one of the topics of research at the instiute. After all, these processes are the foundation for the nerve cells ability to collect and process information including such complex brain functions as learning and memory. The widely ramified extensions of a nerve cell (blue) are scattered with contact points with other nerve cells (red).

2 Synaptic Dynamics and Modulation Nerve cells in our brains communicate at specialized contact sites, the synapses. During the process of chemical synaptic transmission, an electrical impulse leads to the opening of Ca 2+ channels in the nerve terminals of the presynaptic (signaling) cell. The resulting Ca 2+ influx causes the fusion of vesicles with the plasma membrane, and the ensuing release of neurotransmitter from the presynaptic cell. The neurotransmitter in turn activates receptor-ion channel complexes on the postsynaptic (receiving) nerve cell. Interestingly, repetitive electrical activity in the presynaptic nerve cell leads to plastic changes in synaptic strength over short periods of time. The responses in the postsynaptic nerve cell either increase or decrease over time. This dynamic behavior of synapses, or short-term synaptic plasticity, influences the way in which the information flow in neuronal networks is integrated over short time periods. A deeper understanding of the mechanisms of short-term plasticity requires studying the signaling steps in the presynaptic nerve terminal. Such studies are, however, hampered by the small size of most types of nerve terminals. We therefore study an unusually large glutamatergic nerve terminal, the calyx of Held, at which presynaptic whole-cell patch-clamp recordings can be obtained. This allows us to stimulate the nerve terminal electrically in the voltage-clamp mode or chemically by elevating the intracellular Ca 2+ concentration by the photolytic release of caged Ca 2+, caused by a short light flash. The resulting increase in Ca 2+ concentration is measured by imaging the fluorescence of a Ca 2+ -sensitive indicator dye (see image). We expect the accessibility of the calyx of Held nerve terminals to such manipulations to produce new insights into the signaling mechanisms that underlie synaptic transmission and its short-term plasticity. Dr. Ralf Schneggenburger Ralf Schneggenburger studied biology at the Universities of Göttingen and Tübingen, and conducted part of his graduate studies at the University of Seville in Spain. In 1993, he completed his Ph.D. thesis at the Max Planck Institute for Biophysical Chemistry. He then went as a postdoctoral fellow to the University of the Saarland, and to the Ecole Normale Supérieure in Paris, France. In 1996, he joined the Department of Membrane Biophysics at the Max Planck Institute for Biophysical Chemistry. In 2001, he received a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft, and, since then, has been leading the Synaptic Dynamics and Modulation research group. rschneg@gwdg.de Meyer, A. C., E. Neher and R. Schneggenburger: Estimation of quantal size and number of functional active zones at the calyx of Held synapse by nonstationary EPSC variance analysis. J. Neurosci. 21, (2001). Felmy, F., E. Neher and R. Schneggenburger: Probing the intracellular Calcium sensitivity of transmitter release during synaptic facilitation. Neuron 37, (2003). Wölfel, M., and R. Schneggenburger: Presynaptic capacitance measurements and Ca 2+ uncaging reveal sub-millisecond exocytosis kinetics and characterize the Ca 2+ sensitivity of vesicle pool depletion at a fast CNS synapse. J. Neurosci. 23 (2003) A series of fluorescent images of a calyx of Held is used to measure the increase of intracellular Ca 2+ concentration, produced by Ca 2+ uncaging with a short light flash. The right panel also shows the patch pipette used to measure the electrical signals in the presynaptic nerve terminal. departments and research groups 57

3 Neurobiology Professor Reinhard Jahn After studying biology and chemistry, Reinhard Jahn obtained his Ph.D. from the University of Göttingen in Subsequently, he conducted research at the Rockefeller University in New York ( ) and the Max Planck Institute for Psychiatry (presently renamed Neurobiology) in Munich ( ). After this, he was appointed Professor of Pharmacology and Cell Biology at Yale University, New Haven, with a joint appointment at the Howard Hughes Medical Institute. Since 1997, he has been a director at the Max Planck Institute for Biophysical Chemistry and head of the Department of Neurobiology. Reinhard Jahn is also Honorary Professor of the University of Göttingen. In 1990, he received the Max Planck Research Prize, and was awarded the Gottfried Wilhelm Leibniz Prize in Internal research groups: Dr. Dirk Fasshauer Dr. Ulrich Kuhnt Dr. Thorsten Lang rjahn@gwdg.de Neurons communicate with other cells by releasing neurotransmitters stored in synaptic vesicles in nerve terminals. Upon excitation, a few vesicles fuse with the plasma membrane. Like several other groups in our institute, we are interested in the molecular basis of the underlying membrane fusion event. Membrane fusion is not limited to neurons. Every eukaryotic cell is compartmentalized into membrane-bound organelles that are continuously reorganized in a dynamic manner. Membrane-enclosed transport vesicles are generated from precursor membranes and are then transported to their destination where they fuse with intracellular target membranes. Vesicles (green), bound to a sheet of plasma membrane containing SNARE clusters (red) are caught in the act of exocytosis. The vesicles are filled with a peptide linked to a green fluorescent protein. Upon fusion, the peptide is released and disappears. Sutton, B., D. Fasshauer, R. Jahn and A.T. Brünger: Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, (1998). Takamori, S., J.S. Rhee, C. Rosenmund and R. Jahn: Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407, (2000). Fasshauer, D., W. Antonin, V. Subramaniam and R. Jahn: SNARE assembly and disassembly exhibit a pronounced hysteresis (2002) Nature Struct. Biol. 9, (2002). Lang, T., M. Margittai, H. Hölzler and R. Jahn: SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs. J. Cell Biol. 158, (2002). Jahn, R., T. Lang and T.C. Südhof: Membrane fusion. Cell 112, (2003). Most intracellular membrane fusion events are mediated by evolutionarily conserved proteins. Among these, the SNARE proteins are the best candidates for catalyzing the fusion reaction. The first clues to their function were obtained from the study of powerful bacterial toxins, botulinum neurotoxins and tetanus toxin. Although tetanus and botulism affect different neurons, the toxins share a common mechanism of action. They cut SNARE proteins in half, which results in an inhibition of vesicle fusion and thus of neurotransmitter release. SNAREs are abundant on intracellular membranes and readily form stable complexes. Each fusion reaction requires the cooperation of several SNARE proteins. Furthermore, the SNAREs mediating different intracellular fusion reactions are not identical, although some SNAREs participate in multiple fusion steps. According to our working model, the SNAREs operate as»nanomachines«. When membranes get close to each other, the SNAREs engage each other and»zipper up«from their distal ends towards the membrane. Since energy is released during this reaction, the membranes are forced into approximation, thereby initiating membrane fusion. After fusion, the SNAREs have released their energy and need to be 58 departments and research groups

4 Working model showing how SNAREs (red, green, blue) drive membrane fusion by operating as nanomachines that pull membranes into close promixity disentangled with the assistance of chaperone proteins and energy input. Little is known how SNARE proteins are regulated, i.e., how they are activated or silenced. SNARE proteins interact with a long and still growing list of other proteins that regulate their conformation and control their availability for fusion reaction, particularly with regard to regulated exocytosis. Several of these proteins are as essential for fusion as the SNAREs, but for most of them it is not yet known how they function at the molecular level. We aim to find out how the SNAREs manage to fuse membranes, as well as how they are regulated by other proteins. To this goal, we investigate the 3D structures and the conformational changes of SNAREs. Furthermore, we fuse artificial and native membrane vesicles in the test tube. We have also established cell-free but semi-intact vesicle-plasma membrane preparations that retain the capacity for regulated exocytosis. With the help of these approaches, it is possible to learn more about the multiple factors involved in SNARE-mediated membrane fusion. Moreover, we collaborate with the groups of E. Neher, C. Rosenmund, and J. Klingauf to elucidate the effects of molecular changes on exocytosis in intact cells and neurons. Finally, we are also interested in the mechanisms by which synaptic vesicles sequester and store neurotransmitters. Three-dimensional structure of the SNARE complex mediating neuronal exocytosis. The cleavage sites of the different botulinum (BoNT) and tetanus (TeNT) neurotoxins are indicated by arrows. departments and research groups 59

5 Mechanisms of Synaptic Transmission Dr. Christian Rosenmund Christian Rosenmund studied pharmacy at the University of Frankfurt/Main and received his Ph.D. in physiology from the Vollum Institute in Portland, Oregon, in After working at the Salk Institute in La Jolla, California, for two years, he returned to Germany as a Helmholtz fellow in Since 1998, he has been head of the Molecular Mechanisms of Synaptic Transmission research group in the Department of Membrane Biophysics at the Max Planck Institute for Biophysical Chemistry. In 1999, Christian Rosenmund obtained his Habilitation in physiology and is a Heisenberg fellow since then. crosenm@gwdg.de Rosenmund, C., A. Sigler, I. Augustin, K. Reim, N. Brose, and J.S. Rhee: Differential control of vesicle priming and short term plasticity by Munc13 isoforms. Neuron 33, (2002). Rhee, J.S., A. Betz, S. Pyott, K. Reim, F. Varoqueaux, I. Augustin, D. Hesse, T.C. Südhof, M. Takahashi, C. Rosenmund, and N. Brose: β-phorbol ester- and diacylglycerol-induced augmentation of neurotransmitter release from hippocampal neurons is mediated by Munc13s and not by PKCs. Cell 108, (2002). Fernandez-Chacon, R., A. Konigstorfer, S.H. Gerber, J. Garcia, M. F. Matos, C.F. Stevens, N. Brose, J. Rizo, C. Rosenmund, and T.C. Südhof: Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, (2001). Reim, K., M. Mansour, F. Varoqueaux, H.T. McMahon, T.C. Südhof, N. Brose, and C. Rosenmund: Complexins regulate a late step in Ca 2+ -dependent neurotransmitter release. Cell 104, (2001). Many specific proteins are necessary to fill the vesicles and to let them contact and fuse with the cell membrane. The brain is clearly our most complex organ; hundreds of billion neurons process information from the outer world to control along with what we have learned in the past our second-to-second actions. Neurons use specialized junctions, the synapses, for cell-to cell communication. The synapse transduces the electrical activity of the sending, presynaptic neuron via the Ca 2+ -triggered release of neurotransmitter (NT) filled vesicles. The postsynaptic, receiving site transduces the NT signal into an electrical signal via activation of ion channels. Multiple events must occur at the presynapse before NT can be released, because synaptic vesicles need to be filled with NT, tether to specific release sites and be primed to reach fusion competence. We are interested to learn at which stage of release presynaptic proteins act, and how they function. In recent years, we have started to functionally characterize mice bearing deletions of (knockouts), or carrying mutations within presynaptic proteins (knockins). We use cultured neurons to characterize the consequences of these mutations on synaptic transmission with standard patch-clamp electrophysiology. We further study the function of the protein by testing the rescue ability of mutated protein versions when they are overexpressed in knockout mice. Among the examined molecules, we identified and characterized a protein family involved in the vesicle priming (munc13s), and recognized them as key regulators of synaptic short-term plasticity. We also studied mutants of synaptotagmin-1 that confirm its role as a putative Ca 2+ -sensor of release. Neurons lacking the protein family of complexins show reduced NT release efficiency accompanied by desynchronisation of release, indicating an important role of these proteins in Ca 2+ -triggered release. Our long-term goal is to achieve a molecular and functional model of the release process at the central synapse. 60 departments and research groups

6 Microscopy of Synaptic Transmission When nerve cells transmit a signal, this happens in specialized contact zones, the so-called synapses. There, small membrane bubbles, the synaptic vesicles, fuse with the cell membrane and release a transmitter substance. If a nerve cell has something to say, a few dozen of those vesicles will be fused within a few seconds. The membrane needs to be recycled swiftly otherwise the synapse would swell up and the supply of release-ready vesicles would disappear. Thus, the membrane of fused vesicles is invaginated, pinched off, and rearranged in new vesicles that are eventually refilled with transmitter substance. How this recycling process works, however, is not yet understood in molecular detail. Based on electron microscopic investigations, several different mechanisms are being discussed. Such images, however, provide only snapshots of the fast events occurring in living cells. Variants of fluorescence microscopy techniques developed at this institute promise to open up new avenues for viewing these processes in real time. With these tools, we want to spy out some of their dynamic properties. Our objects of study are nerve cells of rat and mouse brain, which we can keep in cell culture for a number of weeks. When placed in culture dishes, freshly isolated nerve cells re-grow and even form new synaptic connections with each other. These we observe dur- Dr. Jürgen Klingauf Jürgen Klingauf studied biology and physics in Hamburg and Bonn. He subsequently spent three years at Stanford University, California, as a Boehringer Ingelheim Fonds fellow, and received his Ph.D. in Physics from the University of Göttingen in Since 2000, he has been head of the Microscopy of Synaptic Transmission group in the Department of Membrane Biophysics at the Max Planck Institute for Biophysical Chemistry. jklinga@gwdg.de ing the entire process, from vesicle transport through fusion with the membrane to vesicle recycling. Although single vesicles are far too small to be visualized by standard light microscopy, their fluorescence signals reveal a lot of vital information. Electron microscopic picture of a synapse packed with numerous synaptic vesicles. Pyramidal nerve cell from rat brain in cell culture. On the right, the small synaptic boutons ( 1/1000 of a millimeter in diameter) are specifically marked with a fluorescent dye. departments and research groups 61