Scientific Focus
Nucleotide signalling, adult neurogenesis
ATP and other nucleotides such as ADP, UTP, UDP, or the diadenosine polyphosphates Ap4A and Ap5A act as signalling substances in the nervous system. Their action is mediated by specific receptors and terminated by hydrolysis via surface-located enzymes (ecto-nucleotidases). The group is studying the molecular biology and functional properties of the various ecto-nucleotidases. Their distribution in the adult and developing nervous system is analyzed using monospecific antibodies and cellular expression is documented by in situ hybridization. More recently we began to analyze the role of ecto-nucleotidases and nucleotide signalling in the control of the formation of nerve cells from stem cells in the adult brain (adult neurogenesis). Interestingly, adult neural stem cells express the ecto-nucleotidase NTPDase2. Activation of nucleotide receptors (P2 receptors) stimulate progenitor cell proliferation synergistically with growth factors. The molecular processes underlying this interaction and their functional consequences for regulating adult neurogenesis are under study using in both, in vivo and in vitro preparations.
Synaptic vesicle proteomics
Neurotransmitter substances are stored in synaptic vesicles and released on stimulation and the gated influx of calcium ions. This process is highly regulated by a molecular machinery that involves synaptic vesicle proteins equally well as soluble proteins of the nerve terminal and proteins of the presynaptic plasma membrane. More recently, we launched a proteomics approach for the identification of novel synaptic proteins from murine brain using immunoaffinity isolation of synaptic vesicles followed by separation of vesicle proteins using one- and two-dimensional polyacrylamide gel electrophoresis and a combination of matrix assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF-MS) and nano-electrospray ionization tandem mass spectrometry (nano-LC ESI-MS/MS). Work aimed at the further functional and molecular characterization of the identified synaptic vesicle proteins is in progress.
Methods
Cell and tissue culture, immunocytochemistry including confocal microscopy, in situ hybridization, general molecular biological techniques, including molecular cloning, PCR, cell transfection, analysis of expressed proteins, general protein biochemical techniques, including immunoaffinity purification, immunoblotting, separation of membrane proteins by one and two dimensional gel electrophoresis (including blue native PAGE, BAC/SDS-PAGE and dSDS-PAGE), preparation of protein sample for mass spectrometric analysis, isolation of neural stem cells, culturing and analysis of neurospheres, in vitro differentiation of stem cells.
Selected Publications
Braun, N., Zhu, Y., Krieglstein, J., Culmsee, C., Zimmermann, H. (1998) Upregulation of the entire enzyme chain hydrolyzing extracellular ATP following transient forebrain ischemia in the rat. J. Neurosci. 18, 4891-4900.
Zimmermann, H. (1999) Nucleotides and cd39: Principal modulatory players in hemostasis and thrombosis. Nature Medicine, 5, 987-988.
Maienschein, V. Marxen, M., Volknandt, W., Zimmermann, H. (1999) A plethora of presynaptic proteins associated with ATP-storing organelles in cultured astrocytes. Glia, 26, 233-244.
Kobayashi, S., Zimmermann, H., Millhorn, D.E. (2000) Chronic hypoxia enhances adenosine release in rat PC12 cells by altering adenosine metabolism and transport. J. Neurochem. 74, 621-632.
Zimmermann, H. (2000) Extracellular hydrolysis of ATP and other nucleotides. Naunyn-Schmiedebergs Arch. Pharmacol. 362, 299-309.
Braun, N., Fengler, S., Ebeling, C., Servos, J., Zimmermann, H. (2000) Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. Biochem. J., 351, 639-647.
Braun, N., Sévigny, J., Robson S. C., Enjyoji, K., Guckelberger, O., Hammer, K., Di Virgilio, F., Zimmermann, H. (2000) Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain. Eur. J. Neuroscience,12, 4357-4366.
Braun, N., Zimmermann, H. (2001) Microglial ecto-nucleotidases: Identification and functional roles. Drug. Dev. Res., 53, 208-217.
Sévigny, J., Sundberg, C., Braun, N., Guckelberger, O., Csizmadia, E., Qawi, I., Imai, M., Zimmermann, H., Robson, S.C. (2002) Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and NTPDase2 have implications for thromoregulation. Blood, 99, 2801- 2809.
Ravi, R.G., Kim, H.S., Servos, J., Zimmermann, H., Lee K., Maddileti, S., Boyer, J.L., Failer, B. U., Braun, N., Zimmermann, H. (2002) Cloning, expression, and functional characterization of a novel Ca2+-dependent ER nucleoside diphosphatase. J. Biol. Chem., 277, 36978-36986.
Braun, N., Sévigny, J., S. K. Mishra, Robson, S. C., Barth, S. W., Gerstberger, R., Hammer, K., Zimmermann H. (2003) Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain. Eur. J. Neurosci. 17:1355-1364.
Ortinau S., Laube, B., Zimmermann, H. (2003) ATP inhibits NMDA receptors after heterologous expression and in cultured hippocampal neurons and attenuates NMDA-mediated neurotoxicity J. Neurosci., 23, 4996-5003.
Braun, N., Sévigny, J., Robson, S. C., Hammer, K., Hanani, M., Zimmermann, H. (2004) Association of the ecto-ATPase NTPDase2 with glial cells of the peripheral nervous system. Glia, 45, 124-132.
Shukla, V., Zimmermann, H., Wang, L., Kettenmann, H., Raab, S. Hammer, K., Sévigny, J., Robson, S.C., Braun, N. (2005) Functional expression of the ecto-ATPase NTPDase2 and of nucleotide receptors by neuronal progenitor cells in the adult murine hippocampus. J. Neurosci. Res. 80, 600-610.
Morciano, M. Burre´, J., Corvey, C., Karas, M., Zimmermann, H., Volknandt, W. (2005) Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis. J. Neurochem., 95, 1732–1745
Mishra, S.K., Braun, N., Shukla, V., Füllgrabe, M., Schomerus, C., Korf, H.-W., Gachet, C., Ikehara, Y. Sévigny, J. Robson, S.C., Zimmermann, H. (2006) Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation. Development, 133, 675-684.
Robson, S.C., Sévigny, J. and Zimmermann, H. (2006) The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance, Puringeric Signaling, in press
Zimmermann, H. (2006) Nucleotide signaling in nervous system development. Pflugers Arch. Eur. J. Physiol., in press.
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