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 (ectonucleotidases) that eventually break down the nucleotides to the respective nucleoside. We are studying the molecular biology, functional properties and cellular distribution of the various ectonucleotidases. More recently we began to analyze the role of ectonucleotidases and nucleotide signalling in the control of the formation of nerve cells from stem cells in the adult brain (adult neurogenesis). Adult neural stem cells express the ectonucleotidase NTPDase2. Activation of nucleotide receptors (P2 receptors) stimulates 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.
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 molecular machinery invovling synaptic vesicle proteins equally well as soluble proteins of the nerve terminal and proteins of the presynaptic plasma membrane. More recently, together with Walter Volknandt, a proteomics approach was launched 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). The work aims at the functional and molecular characterization of the identified synaptic vesicle proteins.
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), isolation of neural stem cells, culturing and analysis of neurospheres, in vitro and in vivo differentiation of stem cells.
Gampe K, Stefani J, Hammer K, Brendel P, Pötzsch A, Enikolopov G, Enjyoji K, Acker-Palmer A, Robson SC, Zimmermann H. (2015) NTPDase2 and purinergic signaling control progenitor cell proliferation in neurogenic niches of the adult mouse brain. Stem Cells 33, 253-264.
Mishra, S.K., Braun, N., Shukla, V., Füllgrabe, M., Schomerus, C., Korf, H.-W., Gachet, C., Ikehara, Y. Sévigny, J. Robson, S.C.and Zimmermann, H. (2006) Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation. Development, 133, 675-684.
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.
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., 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.